(navigation image)
Home American Libraries | Canadian Libraries | Universal Library | Community Texts | Project Gutenberg | Children's Library | Biodiversity Heritage Library | Additional Collections
Search: Advanced Search
Anonymous User (login or join us)
Upload
See other formats

Full text of "Biochemistry"

\9S9 



MmssmaifflFim 




U. 8. NAVAL MEDICAL SCHOOL 
NATIONAL NAVAL MEDICAL CENTEE 
BETHE8DA, MARYLAND 
ltit 






is 

Us 






INTRODUCTION 



This manual was prepared by the staff of the 
Chemistry Branch of the Department of Lab- 
oratories of the Naval Medical School to serve 
as a guide for Laboratory Students and as a 
working reference for both physicians and 
technicians in' the field of Biochemistry. The 
procedures and techniques contained herein 
were selected for their simplicity and accuracy 
and are in current use in the Laboratories of 
the Naval Medical School. 



Jhriifa 



M. W. ARNOLD 

CAPT MC USN 

Commanding 

U. S. Naval Medical School 

National Naval Medical Center 

Bethesda, Maryland 












\P& 






W 



X\\e 












PREFACE 



In recent years clinical pathology has been rapidly 
expanding, especially in the field of clinical chemistry. 
Many procedures which once were considered purely 
research in character, such as transaminase and ster- 
oids have been adapted to the routine clinical labora- 
tory. In addition, there have been many refinements 
and improvements in techniques which have necessi- 
tated changes in time-honored methods. To adequately 
perform laboratory tests it is essential to have a basic 
understanding of sound laboratory principles, such as 
primary standards, methods of analysis, chemical 
balances, etc. These subjects are brought to attention 
in the first part of this manual and will prove extreme- 
ly valuable to medical technologists and residents in 
pathology. The authors are to be commended on the 
completeness and excellence of this manual on Clinical 
Chemistry. 




O 



c 



CAPT, MC, USN 
rector of Laboratories 
S. Naval Medical School 
National Naval Medical Center 
Bethesda, Maryland 






ii 












This Manual, in its original form and subsequent 
revisions, is the result of the combined efforts of 
both former and present members of the Staff of the 
Naval Medical School. 



" most human activities advance 

by virtue of contributions from many dif- 
ferent types of individuals, with vastly 
different endowments, working at differ- 
ent levels. Medical investigation if no 



exception to this rule. " 












Medical Research: A Mid- 
Century Survey 
Boston, Little, Brown and 
Company, 1955, vol. 1, p, 
xxxi. 






■ 









Hi 






TABLE OF CONTENTS 



G eneral Chemical Tcchnlc 

Introduction , l 

Organization and Control in the Laboratory 1 

Laboratory Discipline , , 2 

The E mergency in the Laboratory 2 

Methods of Analysis , 3 

General Chemical Technlc 4 

Chemical Balances 4 

Volumetric Analysis 11 

General Instructions for the Preparation of Solutions . . 19 

Primary Standards 20 

Secondary Standards 27 

Volumetric Calculations * 32 

Acids , Bases and Buffers 39 

Colorlmetric Technics 48 

Photometric Technics , , 53 

Gasometric Analysis 61 

Urine Collection and Preservation 67 

Feces Collection and Preservation 68 

Collection of Blood Specimens . , . . 70 

Analytical Methods 

Protein-free filtrates 76 

Acetone 83 

Adrenal Insufficiency Test 85 

Alcohol, ethyl 88 

Alcohol, methyl , 91 

Amino Acids 93 

Amylase 

glucose method 97 

starch-iodine method 101 

Ascorbic Acid 103 

Ascorbic Acid Saturation Test , 106 

Bilirubin 107 

Bilirubin (micro-method) 109 

Bromide < . . . 110 

Bromsulfaleln Tolerance 112 

Bromsulfaleln Clearance , 115 



lv 



( 



TABLE OF CONTENTS (cont.) 



Analytical Methods (cont.) 
Calcium 

oxalate mctlwd „ 118 

chloranllic acid method 127 

Carbon Dioxide 

volumetric method - 129 

manometric method 137 

Carbon Monoxide 140 

Cephalln Flocculatlon ...... 144 

Chloride 

mercurlmetric . . . . , 145 

argentimetrlc-adsorption Indicator t 147 

argenttmetrlc-thiocyanate Indicator 148 

Cholesterol 

Bloor , 152 

Schoenhelmer and Sperry ... •#.... 164 

Congo Red Test i 158 

Creatinine 

Folin-modifled 159 

Lrfken-modified 160 

Creatine 162 

Esterase (tributyrinaae) 164 

Estrogens 168 

Fat (in feces) ■. 171 

Fibrinogen 

clot time -. 175 

turbldimetrlc 177 

Follicle Stimulating Hormone (FSH) 180 

Glucose 181 

Glucose Tole ranee ............ 183 

5-Hydroxy-indoleacetic acid (serotonin derivative) 185 

Icterus Index 187 

Iodine , protein-bound ■ 188 

Iron 195 

Iron binding capacity 198 

17-Ketosteroids (neutral) * 201 

Lipase (of pancreatitis) 207 

Magnesium 

titan yellow method 209 

phosphate method 211 



TABLE OF CONTENTS (cont.) 



Analytical Methods (cont.) 

Nitrogen (macro-kjeldahl) 213 

Nitrogen (mlcro-kjeidahl) 215 

Phosphatase 

King- Armstrong-modified 218 

Bodansky-modified 225 

Phosphorus 

Inorganic 224 

lipid 228 

Porphobilinogen k 229 

Porphyrins 

qualitative 230 

quantitative 233 

Potassium (and Sodium) 4 250 

Protein 

turbidlmetric l 236 

biuret -tartrate 237 

bturet-EDTA 239 

Prothrombin time 

plasma , 240 

serum „ 248 

Salicylic Acid 249 

Sodium (and Potassium) , 260 

Sulfonamides 254 

Thiocyanates 256 

Thymol Turbidity 257 

Transaminase 258 

Urea 

Boutwell method 264 

Karr method 268 

Uric Acid 271 

Zinc Turbidity (gamma globulin) 273 



Toxicology 






Introduction 274 

Reinsch Test for Heavy Metals 277 

Arsenic » . • 279 

Mercury 282 

Lead 284 



Vl 



I 



TABLE OF CONTENTS (cont.) 



Toxicology (cont.) 
Barbiturates 

quantitative 287 

qualitative-paper chromatography 292 



Appendix 



Simple Blood Tests for the General Practitioner , 294 

Routine Chemical Analysis of Urinary Calculi 298 

Choice of Laboratory Tests 304 

Clinical Interpretation of Laboratory Tests (table) 306 

Sample Size Requlredior Analysis (table) 313 

Control Procedures in the Clinical Chemistry Laboratory ........ 315 

Statistics 317 

Acid Base Equilibrium and Water Balance 321 

Coagulation of Blood 324 

Turbidity Standards 329 

Calibration of Photometer Tubes 330 

Volume Calibration of Test Tubes » » 332 

The Use of the Slide Rule 333 

Common Logarithms of Numbers (table) 334 

Optical Density vs. Transmittance (table) 336 

Standard Reference Texts -..«,« 340 

Bibliography 341 

Index 350 

Periodic Classification of the Elements 3S9 

Table of International Atomic Weights 359 



vii 



ILLUSTRATIONS 



Figure Page 

1 Analytical Balance 5a 

2 Prescription Balance 6a 

3 Trip Balance , , . . . 6a 

4 Triple Beam Balance 6a 

5 Solution Balance . . . . j 6a 

6 Volumetric Flask 13a 

7 Transfer plpet 14a 

8 Measuring plpet • 14a 

9 Serological plpet 14a 

10 Ostwald-Folin plpet 14a 

11 Capillary plpet 14a 

12 Folln-Wu plpet 14a 

13 Buret 14a 

14 Comparator Block ..»*•■ 44a 

15 Duboscq colorimeter 48a 

16 Colorimeter principles 48a 

17 Simple filter photometer and 

spectrophotometer 53a 

18 Graphs 54a 

19 Absorption and trausmlttance 

spectra 54a 

20 Thermometer scales 62a 

21 Bogen 1 s apparatus 88a 

22 Bromsulfalein clearance 115a 

23 Van Slyke-Cullen Volumetric C0 2 

apparatus 132a 

24 Carbon monoxide analysis 140a 






( 



viil 









JNTKODUCTION 



This manual, in general, represents the methods in uiie at the present time In 
this labor.itory. Some methods in use in the author's laboratory in 195G (Temple 
University Hospital, Philadelphia, Pa.) are included as alternate procedures. 

These methods derive from many sources, most of which are referred to in the 
reference section for each procedure. Commonly, the exact procedure is slightly 
modified to accord with the requirements of this laboratory. 

The sections on general principles of chemical analysis will, It ly hoped, serve 
to help to encourage accurate, precise, and knowledgeable work in the laboratory. 
The material on interpretation should serve as a motivating link between the labora- 
tory worker and the physician in their joint effort directed toward the diagnosis and 
treatment of disease. 

There are, undoubtedly, errors present in this manual, in typography and in text. 
It is hoped that these will be drawn to our attention so that they may be rectified. 

Clinical chemistry seeks by the analysis of biological fluids and tissues to aid 
the physician in the diagnosis and treatment of disease. The laboratory cannot diag- 
nose but it can aid in diagnosis; the laboratory cannot treat but it can help to guide 
the physician in his therapeutic efforts. The laboratory Is responsible for prompt, 
rapid and accurate chemical analysis of specimens submitted to it for examination. 
In many cases, the physician can aid the laboratory by supplying certain Information 
about the patient, such as previous pertinent medical history. 



ORGANIZATION AND CONTROL IN THE LABORATORY 



A clinical chemistry laboratory should be so staffed and organized that the inevi- 
table errors of technique and of human fallibility will be immediately recognized bo 
that steps may be taken to insure that: (a) the erroneous result will not be sent to 
the physician and (b) the source of the inaccuracy can be found. 

Each individual must be alert to the possibilities of error and when a mistake is 
detected, must be willing and eager to repeat the determination. A laboratory should 
never be so under pressure from excessive work load that adequate measures for in- 
suring the requisite degree of accuracy are neglected. It Is in evitable that errors In 
solution preparation, analytical technique and In the calculation of results shall occur; 
it must be made inevitable that these errors be detected an d remedied before they are 
translated into tragedy for the patient. It can never be as serious for an analyst to 
admit a mistake as it is for the patient to experience the results of the mistake I 



Therefore, It should be evident that the analyst must be completely honest In 
the obtaining and the calculation of data, not only with hla fellow -workers, but also 
with himself. This Is always difficult; the one person easiest to deceive Is one's own 
self. Always remember: your technique may be perfect, the method or solution may 
be at fault. 

In this laboratory, results are never given to any person other than those in im- 
mediate charge of the care of the patient: the doctors (the attending physicians) and 
the nurses. Any others desiring information should secure it from the physician in 
attendance. Also, the interpretation of results in the laboratory should not be made 
by the technician except by repeating printed values of normal ranges which are avail- 
able. Remember — the laboratory cannot diagnose I 

The next few paragraphs are taken from "A Curriculum for Schools of Medical 
Technology" by Israel Davidsohn, M.D, , who is Director of Laboratories and Path- 
ologist at Mount Sinai Hospital, Chicago, Illinois. 



LABORATORY DISCIPLINE 

It should always be remembered that any consideration of medical technology Just 
as of medicine in general must be approached from the viewpoint that the final goal of 
all work is service to the patient. The patient Is the center; everything in medical 
work — and medical technology Is a part of it — revolves around the patient. 

This makes it easy to understand the need for discipline in the medical laboratory, 
which can be likened to a ship on the ocean. Just as the captain of the ship cannot be 
held responsible for his task unless there is rigid discipline, so must there be dis- 
cipline in the laboratory if the pathologist is to do well his part of the medical service 
to the patient. 

The technologist must realize the essential need of laboratory discipline; he must 
be ready and willing to adapt himself to the organization of the laboratory) although It 
may sometimes entail personal hardship and Inconvenience. Those who cannot or will 
not do it should keep away from medical work of any kind. 



THE EMERGENCY IN THE LABORATORY 



There are occasions-, rather frequent ones, when the life of a patient depends on 
the results of laboratory tests, on their accuracy, on the speed with which they are 
performed and on their interpretation. The Interpretation is entirely within the scope 
of activities of the pathologist, as has been stated already. The accuracy of the results 



2 



( 






and tire speed of performance are two aspects of laboratory work for which the medical 
technologist in responsible. In his training these two features muat always be remem- 
bered and emphasized . Medical technologists must be trained in school and long after 
they have completed their undergraduate education — to be ready to face laboratory 
emergencies with knowledge, with mastery of technic, with a cool mind and solid 
Judgment, and with a readiness to serve at any hour of the day or night, on Sundays 
and holidays, regardless of inconvenience and hardship. 

This is a part of the medical ethics of the medical technologists In its broad con- 
cept, Just as it is a part of the medical ethics of the physician. 






METHODS OF ANALYSIS 



This manual presumes that the laboratory technician has a basic knowledge of gen- 
eral chemistry and its laws. The Handbook of the Hospital Corps, U. S. Navy, 1953, 
contains an excellent section on the basic laws of chemistry. The special basic chemi- 
cal principle of each Individual determination is outlined as each method is presented 
In the manual. 

There are four general methods in use in chemical analytical laboratories. These 
are: 

1. Gravimetric methods In which the estimation Is made by weighing an Isolated 
purified substance, which may have been originally present in the sample or formed 
by a reaction. 

2. Volumetric (titrimetric) methods which are based on the measurement of the 
volume of a reagent used up during the reaction with a measured amount of sample. 

3. Colorlmetrlc methods in which the amount of colored material present or 
formed by a reaction Is measured by light absorption techniques. 

4. Gasometric methods in which the amount of a gas present or produced by a 
reaction is measured either by 

a. Measuring the volume at known conditions of temperature and pressure 
and water vapor saturation or 

b. Measuring the pressure at known volume, temperature and water vapor 
saturation . 

The apparatus commonly used In these gasometric methods for clinical pro- 
cedures are respectively (a) the Van Slyke volumetric and (b) the Van Slyke mano- 
metric apparatus. 

Most of the methods used in laboratories today are colorlmetrlc, one or two are 
gasometric, a number are volumetric and almost none are gravimetric. 



Aii analytical chemical method to bo used In a routine clinical chemistry labora- 
tory must meet the following requirements: 

1. It must be accurate enough and precise enough for clinical Interpretation. 

2. It must require a sample small enough to be obtained without harm to the 
patient. 

3. It must give results In a short enough time that they become clinically useful. 
A rapid method often is of much greater value to the physician (and to the patient!) 
even though it may be less precise than a slower one. Every laboratory of necessity 
often chooses reluctantly between accuracy and speed and accepts the compromises 
inherent In such a choice. 

Volumetric and Colorlmetric methods will be discussed further here. Gravimet- 
ric methods will not be discussed further as a method of analysis, but since the weigh- 
ing of materials in the preparation of solutions is a very large and important part of 
laboratory work, the use of laboratory balances will be further discussed below. 



GENERAL CHEMICAL TECHNIQUE 

Quantitative analysis differs from qualitative analysis not only in its ultimate ob- 
jective but In the details of manipulation of the analysis. Quantitative analysis requires 
great care to achieve the precise measurements necessrry to accurate determinations; 
whereas, in qualitative tests, rather large errors In measurement affect the results 
only very slightly. 

The recommendations and general rules which will be noted here are for the pur- 
pose of increasing the accuracy and precision of the individual methods collected 
herein. The acquiring of skill in chemical technique is much like learning to drive a 
car or to play the piano. The purpose Is to achieve accuracy In a chemical determina- 
tion and rules are set up to help to do this. Also, like learning these other skills, the 
time will come when the separate rules are forgotten but the integrated technique 
remains. But bad habits as well as good ones can be learned, so at the beginning of 
your instruction, follow to the letter the rules which are outlined for you; always, of 
course, looking for the reason behind the rule. 

CHEMICAL BALANCES 



The determination of the weight (mass) of a substance is one of the fundamental 
physical measurements. In clinical chemistry, a number of different types of 



( 



( 









balances are used depending upon (1) the accuracy required In the measurement and 
(2) the total mass which la to be weighed. 

These two factors are somewhat Into r-related — the most sensitive balances being 
designed to handle only very limited loads— the less sensitive being designed to handle 
greater loads. It is possible, but difficult, to design and construct a balance to be 
both highly sensitive and able to handle great loads (large capacity). 

Types of Balances 

It is important to choose the type of balance required for a given Job. If we de- 
sire to weigh out 10 g. of material with a required accuracy of only 0. 5 g. then we 
should use a trip balance, accurate to about O.lg,; the analytical balance should not 
be used since its accuracy is excessive (0.0001 g-). 

1. The analytical balance (Figure 1, p. 6a). This is the most sensitive of the com- 
monly used balances. It is used in the preparation of standard solutions and wherever 
accuracy to one milligram or less is required. The load capacity is about 100-200 g. 

2. The prescription balance (Figure 2, p. 6a). This type of balance has a load 
capacity of about 100 to 200 g. and is accurate to about 10 mg. (0.01 g.) It is common- 
ly a torsion type balance — depending on the twisting of a wire or a steel tape — and con- 
taining no knife edges. Its relatively great sensitivity also requires (as does the anal- 
ytical balance) that the pans be protected against air currents during weighing. 

3. The trip balance (Figure 3, p. 6a). This is a most useful balance. It has a 
load capacity of about 2000 g. with a sensitivity of about 100 mg, (O.lg.). It Is 
equipped with separate weights and a sliding beam weight or with an adjustable beam 
weight plus -a slider. 

4. Triple -beam balance (Figure 4, p. 6a). This is a very convenient type of bal- 
ance for general weighing in the laboratory with a capacity up to about 200 g. and a 
sensitivity of about 10 milligrams. It has only one pan, attached to the short arm of 
a beam with arms of unequal length. On the long arm, the beam has three graduated 
weight scales located In a single horizontal plane, each carrying its own attached 
rider, two of the scales are notched and the third is equipped with a sliding rider. 

5. The solution balance (Figure g, p. 6a). This is of use where large volumes 
of solutions are being prepared. It is equipped with a sliding beam weight for taring 
a beaker or other container, as well as separate weights and sliding beam weights. 
It has a capacity of 20,000 g. (20 kilograms) with a sensitivity of 1000 mg. (lg,). 

There are numerous special types with varying capacities and sensitivities for 
special uses. 







( 






( 






Figure 1. Analytical Balance 



6a 



Care and Maint en ance o f Bala nces - General Principles 

1. Balances are, In most instances, constructed with a rigid horizontal beam 
(or lever) with the two ;irms of equal length {except In the case of the triple beam (4) 
and the solution balance (5) above) , with two pans suspended at the ends of the beams 
on knife edge supports. The center of the beam, the fulcrum, Is another knife edge 
lying In the same plane but above the center of gravity of the system. 

2. These knife edges and the plane surfaces on which they rest during the weigh- 
ing operation are the most vulnerable points In a balance. The balance should always 
be treated with gentle care so as to protect these points of strain. 

Analytical and triple beam balances have devices which allow the knife edges and 
the plane surfaces on which they rest to be separated very slightly whenever objects 
are placed on or removed from the pans. During times when the balance Is not In use, 
the knife edges should be lifted from the plane bearing surface to prevent wear. The 
beam should always be lowered slowly and" carefully. 

3. Most balances must be level for accurate work and are equipped with plumb bobs 
or Spirit-levels of some description which should be checked before any weighing is done, 

4. Balances should be located away from exposure to direct heat such as radiators 
or direct sunlight and should not be exposed to blasts from fans or other sources of 
rapidly moving air. 

All objects must be at the temperature of the balance room before they are weighed. 
Warm or cold objects produce currents of air In the balance case which interfere with 
accurate weighing. 

5. Substances should never be weighed directly upon the pans of any balance. In- 
stead, special "weighing paper" or tared watch glasses or beakers should be used. 
The balance should never be exposed to corrosive fumes. H possible a separate room 
should be used to avoid this. When corrosive substances must be weighed, they should 
be In a closed weighing bottle. 

Special Precautions for the Analytical Balance 

1. Never place on or remove from the balance pans any weight or vessel without 
"arresting" the balance ; that is, raising the mechanical supports so that the knife 
edges no longer touch their bearing surfaces. 

2. The beam and pans must be released gently to avoid injury to the knife edges. 

3. The balance must never be left with the case open or with the beam unsupported. 
The rider should be left at the zero position. 

6 







$ 




w> 




ei 



Pm 







1 



N 









4, Dc careful to avoid spilling materials on the pans or on the floor of the balance 
case. If this happens, remove at once by dusting carefully with a camel's hair brush. 

5. Never set on the pan any vessel having moisture or chemicals on its outside 
surface. Corrosion may otherwise ensue. 

Operation of Balances (other than the analytical balance) 

There is usually some device to indicate the neutral or zero point of the balance. 
The indicator should be centered on the zero point at the beginning of weighing and at 
the end of the weighing. 

Problem : To weigh out 10.5 g. sodium chloride, using the trip balance. 

1. Make sure that the balance is level and the adjustments are made so that with- 
out any weight on the pans on either side that the indicator is at rest at the zero point. 

2. Place a clean dry watch glass on the left pan. Now using the tare beam, or a 
similar watch glass and glass beads placed on the right pan, bring the balance back to 
indicate the zero point. At this point, the container is said to be "tared." 

3. Now place weights totaling ten grams on the right pan, and adjust the beam 
scale rider to read 0. 5 g. (In some balances there are two beams with a large and a 
small rider to eliminate the use of loose weights.) 

4. Add sodium chloride, by spatula, to the watch glass on the left pan* until the 
indicator again reads zero, indicating that there Is now 10.5 g. of NaCl present. 

5. Each balance has its special devices and these should be noted for the most 
efficient use of each instrument. 

Care of the Weights 

An instrument of extreme precision is of little use without a set of accurate weights 
The weights should be just a shade more accurate than the sensitivity limit of the bal- 
ance, with which they are to be used. There is little point in using analytical grade 
weights on a trip balance. Conversely, there is little poirt in using crude weights with 
an analytical balance. 

Analytical (and indeed all) weights should be treated with great respect and with 
intelligent care. They should be always kept In a suitable box, each weight in its own 
proper place. The weights should be removed from the box or from the pan of the bal- 
ance with a set of forceps, never with the fingers. They should never be left on the 
balance pans. If a particular weight has lost its polish or lustre do not attempt to res- 
tore it. In many sets of weights the following values are found: 



50 g., 20 g., 10 g., 10» g., 5 g., 2g., 2' g., i g„ 500 mg., 200 mg., 100 rag.. 100' mg. , 
50 mg., 20 mg., 10 mg,, 10 1 mg,, and a rider equivalent to :i 10 mg. welglil. The rider 
is usually a piece of platinum or aluminum wire looped bo that It rents securely on the 
graduated beam and can be moved conveniently by a special rod provided for the purpose. 

Weighing by Swings 

Determination of the zero point : The pans and then the beam are carefully re- 
leased, so as to allow the beam to swing freely, with an excursion of the pointer of 
about five or six divisions from the midpoint of the scale. Readings are then taken 
of the extreme points on the scale reached by the pointer In swinging — the "turning 
points." Two readings are taken on one side of the middle line and one on the other. 
The first one or two swings are always neglected. 

Example : After the pointer returned from its swing to the left, the following 
"turning points" were recorded: 

Left Right 

6. divisions 
5. 2 divisions 5. 6 divisions 



The turning point on the right corresponding to the 5. 2 on the left is the mean of 
the two readings on the right, — or 5. 8 divisions. Note: that in this case, the turning 
points are not equidistant from the middle point. (If they had been, the zero point would 
have been at the middle point of the scale. ) ff magnitudes to the left are given a nega- 
tive sign and to the right a positive sign, the true rest point is their algebraic sum 
divided by two; thus: 

~ 5 - 2 / 5B = /0. 3 divisions 
2 

The zero point Is thus 0. 3 divisions to the right of the middle line. This is the point 
at which the pointer would stop if it were allowed to come to a stop undisturbed. 

This same procedure is used in actual weighing to determine the exact tenth of 
a milligram (see below). 

Wei ghing by swings: In weighing on the analytical (or other) balance the substance 
or weights can be added or removed until the original zero point is re-attalned. Thiq 
is a tedious process when weighing to tenths of a milligram. Much time ran be saved 
by estimating tenths of a milligram by noting the amplitude of the Bwings as above; 
Weight is added to the right pan and the rider adjusted until the pans are balanced 
within one milligram. The rest point is then determined as above for the zero point. 



< 



I 






The posillon of the rider 1b then shifted one milligram and the rest point Is once more 
determined. The weight at the zero point Is now determined by calculation as Illustra- 
ted in the following example: 

A coin (a U.S. nickel) was placed on the left pan of a balance with a zero point 
(previously determined as above) of / 0.3 (the plus notation is used for values to 
the right of the midpoint of the scale). Weights placed on the right pan amount to 4. 99 g. 
and the rider Is located at 8 milligrams to bring the balance within 1 milligram of equili- 
brium. With the weights totaling 4.998 g. the rest point is now determined to be located 
at /1.5 divisions. (1.5 divisions to the right.) 

The rider is now shifted to the 9 milligram point on the beam. With the weights 
now totaling 4.999 the rest point is found to be located at -0.8 divisions (0. 8 divisions 
to the left) . 

Our data are therefore as follows: 

Zero load - zero point is /0.3 

Total weight 4. 998 . the rest point Is / 1. 6 

Total weight 4, 999 , the rest point is -0.8 

Calculation : One milligram produced a shift from / 1.5 to -0.8, which is 2.3 
divisions; that is, a change of weight of 1 milligram is equivalent to 2.3 divisions. 
This value is known as the sensitivity , and varies slightly with the load. The zero 
point does not vary with load. 

The additional weight needed to move the pointer from a rest point of / 1. 5 to /0. 3 
is the weight needed to move the pointer 1.2 scale divisions. / 1. 5 - {/ 0.3) = 1.2 
Since each milligram moveB the pointer 2.3 scale divisions then 1. 2 m n.52 nig. re- 

2.3 
required to bring the rest point to exactly /0. 3, 

Therefore, the nickel weighs 4.998 / 0.00052 = 4.99852 g. 

The calculation can also be made from the other direction as follows: 
Total weight 4. 999 rest point -0. 8 
zero point /0. 3 

The weight must be reduced sufficiently to change the rest point from -0.8 to /0.3 
A change of 1. 1 scale dMsions. 1.1/2.3 = 0.49 mg. 

Therefore the nickel weighs 4.999 - 0.00049 = 4. 99851 g. 

Note : 

When a precise amount of a chemical must be weighed out, It is advantageous to 
use a prescription balance to approximate the amount needed. The weighing may then 
be completed precisely on the analytical balance. 



Damped Balances 

Some balances are equipped with dampers, cither air cylinder or magnetic so that 
the oscillation may be reduced to one or two swings. This makes It possible to read 
the rest point directly instead of uslnp, the calculations Indicated above. Otherwise the 
procedure Is essentially Identical. 

Chain-O-Matic 

Some balances are arranged so that a hanging chain with a variable loop size can 
be used as a substitute for the rider. These devices speed weighing at very little sacri- 
fice in accuracy and precision. There are many variations In the application of the 
rider principle and supplementary weights. Each balance should be studied before use 
to obtain the greatest advantage from these devices. 

Calibration of Weights 

For the greatest accuracy, the weights used with an analytical balance should be 
calibrated so that corrections for the inaccuracies can be made. Details of this pro- 
cedure can be found in books on quantitative analysis or in the following articles: 

Richards, T. M. , J. Amer. Chem. Soc. , 22, 144 (1900) 

Blade, E. , Indust. and Eng. Chem., Anal. Ed., 11, 499 (1939) 

~ ( 

Alnsworth, A. W. , Indust. and Eng. Chem, Anal. Ed, , 11 , 672 (1939) 

Craig, A., Indust. and Eng. Chem. , Anal. Ed. , .11, 681 (1939) 



10 






VGLUMtiTUIC ANALYSIS 



Volumetric definitions : 

^ mol^r solution of any chemical compound Is a solution oT such concentration that 
one liter contains one gram molecular weight of the compound; e.g. , the molecular 
weight of HCl Is 36.46 g. A molar solution of IICl contains, therefore, 36.46 g. of 
HC1 per liter; a molar solution of H 2 S0 4 (Mol. Wt. 98.08) contains 98.08 g. per liter 
of solution. 

A normal polutlon of any compound Is a solution which contains one gram atomic 
weight (1.008 g. ) of reacting hydrogen per liter, or one which can quantitatively re- 
place or react with an equal volume of such a solution. The fraction of a gram-mole - 
cular weight in a liter of normal solution (containing a gram -equivalent weight) de- 
pends upon the reaction for which the substance is used. A normal solution of H 2 S0 4 , 
with a molecular weight of 98.08, contains 49.04 g. per liter, because of the presence 
of two reacting hydrogen atoms per molecule. 

In acid-base (alkali) titrations, a normal acid solution contains per liter the amount 
of acid which has 1 gram atom of hydrogen replaceable by alkali (base) at the pH used 
as an end point in titration. 

A normal alkali solution is one which neutralizes, volume for volume, a normal 
acid solution. A normal solution of NaOH Is molar but a normal solution of Ba(OH) 2 
Is half molar. 

In oxidation-reduction reactions, a normal reducing solution Is one of which a liter 
contains 1 gram atom of oxidizable hydrogen or its equivalent in other reducing sub- 
stances. Oxalic acid, HgC-O., has two hydrogen atoms, both of which are titratable 
with alkali, and both of whfch are oxidizable by permanganate. Thus a normal solution 
of oxalic acid whether for acidimetry or for oxidation by permanganate, Is half molar. 

A normal oxidizing solution is one of which a liter will oxidize 1 gram atom of 
hydrogen, or Its equivalent of other reducing substances. 

Volumetric apparatus and its use : 

In volumetric analysis, cylinders, flasks, pipets and burets and other glassware of 
various types are used. These consist of containers very accurately graduated either 
"to contain" or "to deliver," very accurately, a certain volume of solution, usually 
aqueous. Measuring cylinders are graduated in milliliters and are used for measure- 
ments where a high degree of accuracy Is not required, and when properly used, are 
suitable for many measurements. For good work, one should always attempt to obtain 
the greatest accuracy possible with the instrument at hand. All apparatus for accurate 
work should be calibrated by the chemist himself. 

11 



In order to obtain satisfactory results in the measurements of liquids In such 
vessels. It is necessary to learn to read the meniscus properly. With transparent 
solutions the bottom of the meniscus Is read; with deeply colored and non-trans- 
parent solutions, such as strong permanganate solutions and blood, the reading Is 
made at the lop of the column of fluid. In making a reading of the volume in a buret 
or flask, it will be found that as the eye is raised or lowered the apparent position 
of the liquid meniscus alters. To avoid such errors (parallax) the reading must be 
made with the eye at the level of the top of the column of fluid. Most vessels have 
graduations which completely encircle the tube or neck to help eliminate errors due 
to parallax. 

Cleaning of apparatus: 

Detergents : There are many non-ionized, synthetic detergent products available 
which do an excellent Job of cleaning glassware. Their efficiency is increased many- 
fold If used hot. There is a tendency to use too concentrated a solution; about a 1% 
solution of most products works very well. 

"Cleaning solution" : One of the most efficient solutions used for the cleaning of 
volumetric glassware Is composed of various concentrations of sulfuric acid satura- 
ted with a dichromate salt but it must be handled with the greatest of care since it is 
an oxidizing acid solution and attacks and destroys skin, clothes, desks, floors and 
books, etc. It should be chosen for use only after detergents have been tried and 
found wanting. 



I. K 2 Cr 2 ? 100 grams 

Concentrated sulfuric acid (tech.) . . , 250 ml. 
Water , 750 ml. 

Note: The concentrated sulfuric acid should be poured Into water. 

n. Na Cr 2 7 .2H 2 200 grams 

Water 100 ml. 

Dissolve, cool. Pour into this solution with continual stirring 1500 ml. 
of concentrated H 2 S0 4 (tech. grade). This cleaning mixture is strong 
and more dangerous and should be used only with dry glassware since 
salt precipitates out if it is diluted with water. 

The purpose of special cleaning procedures for volumetric glassware is to main- 
tain the apparatus free from grease which when present causes the surface of the glass 
to be "non-wetting" and droplets of aqueous solutions then will collect on the surface 
rather than spreading in the thin film which is essential to accurate results. If the 
failure to "wet" iA due not to grease but to "silicones" the problem of removal be- 
comes more difficult. The use of alcoholic KOH overnight may be helpful. 



12 



( 









After use of any of the above, the cleaned apparatus should be rinsed many times 
with tap water and finally several times with small amounts of distilled water. During 
the rinsing, the fingers should be kept off the cleaned inside surfaces which are of vol- 
umetric importance — fingers are greasy and will easily contaminate the cleaned glass- 
ware so as to require re-cleanlng. 



Drying of apparatus : 






Sometimes it is desirable to dry the apparatus quickly. This can be done (a) by 
rinsing with alcohol, followed by ether, or (b) by rinsing with acetone or (c) by heat. 
In any case, use of a current of air hastens evaporation. Note, however, that these 
procedures are desirable only when the apparatus must be dry. Usually it is better 
Just to rinse the apparatus with some of the solution to be measured, If sufficient sol- 
ution is available. Rinse several times with small amounts of solution, allowing good 
drainage between rinses. Three rinsings with 5 ml. are better than two with 10 ml. , 
or one with 30 ml. When air is used for drying it must be clean. A drying oven Is 
very convenient for large numbers of apparatus but is somewhat slow. It must be set 
at a temperature above the boiling point of water for useful application. It has recent- 
ly been shown that heating pyrex volumetric apparatus (volumetric flasks and pipets) 
to very high temperatures (300°C.) does not impair their accuracy to a degree notice- 
able In the clinical chemistry laboratory. J. Chem. Ed. 33, 609 (1956). 

Common volumetric glassware : ■ (See Fig. 6-13, pp. 13a, 14a) 

1. Graduated cylinders are relatively inaccurate measuring devices and are used 
for situations in which accuracy of a high order is not required. They can, however, 
be used very conveniently for the measuring of 24-hour urine volumes of 500 to 2QO0 
ml. where the accuracy afforded is quite sufficient. 

2. Volumetric flasks are designed to allow accuracy to one part in a thousand 
(0. 1%). They are flat-bottomed, pear-shaped vessels with long narrow necks. The 
U. S. Bureau of Standards has issued bulletins outlining the requirements which they 
recommend for the highest accuracy and most manufacturers comply with these rec- 
ommendations. The long narrow necks make it possible to adjust the final total vol- 
ume with ease and precision. The neck is encircled with a thin line indicating the 
capacity of the vessel to that mark, at some definite temperature usually 20°C. In 
volumetric flasks the mark usually indicates the "contained" volume. In some older 
German flasks two marks have been made, indicating a "to deliver" and a "to contain" 
calibration. Most flasks of recent origin have only a "to contain" calibration. 

Volumetric flasks are used to prepare solutions of substances so as to have a 
certain known concentration. The required (calculated) amount of the substance is 
weighed or measured out and transferred to the flask, water is added to about 1/2 of 
the volume of the volumetric flask. The substance is completely dissolved, the vol- 
ume is made up to the mark, avoiding parallax and completely mixed. It is good prac- 
tice to invert, and mix at least ten times after bringing to volume. 

13 





Figure 6. Volumetric Flask 



( 



< 






13a 



Mild heat may be used in bodic c:ihch to speed the dissolving but the final adjust- 
ment to the mark should be made only after cooling to the calibration temperature. 

In no case should the prepared solution be stored in the volumetric flask since 
they are subject to breakage and etching. Volumetric flasks are not inexpensive, 

3. Pipets are cylinders drawn out at one end to form a delivery tip and at the 
other to form a mouth-piece (since the pipet is usually filled by mouth suction). They 
are graduated at a single mark on the upper limb of the pipet to fix the volume of 
fluid which will be delivered under certain specified conditions. Some plpets have 
many subdivisions and are employed for measuring out variable amounts of fluid. 
These are of lower accuracy. 

a. Transfer pipe ts (Fig. 7, p. 14a) are calibrated to deliver a certain vol- 
ume of fluid. The accuracy possible is of the same order as volumetric flasks (0. 1%). 
In order to attain this accuracy the orifice must be of such a Bize that the outflow of 
the fluid Is not too rapid for otherwise slight differences In drainage time give large 
variations in delivery volume. The Bureau of Standards has also set up requirements 
for this type of pipet. 

b. Measuring pipets (Fig. 8, p. 14a) or "Mohr" pipets consist of a cylinder of 
uniform bore calibrated with multiple graduations; they have a lower order of accur- 
acy and are made to deliver "between marks," That is, they are not allowed to drain 
to the tip but the measurement is made between two marks on the pipet. 

c. Serological pipets (Fig. 9, p. 14a) are not as accurate as transfer pipets and 
are used when their margin of error will not affect the results of the determination. 
Both the measuring pipet and the serological pipet are graduated assuming that the 
Internal bore is of uniform diameter and this is not always true. Titrations should 
never be made using measuring or serological pipets. They are usually "blow-out" 
pipets and are designed to deliver as noted below under Ostwald-Folln type. 

d. Ostwald-Folln pipet (Fig. 10, p. !4a) Is a pipet especially designed with a 
large oval bulb and a short delivery tip so as to minimize the effects of viscous sub- 
stances In the measurement of the fluid volume. It is used in the measurement of 
blood, serum or plasma. It Is calibrated to deliver "by blow-out." That is, the pipet 
Is allowed to drain to the tip and then the small amount of fluid that remains in the 

tip after drainage is expelled by closing the upper end with the forefinger and warming 
the bulb by gripping it with the palm of the other hand. The warmed air expands and 
forces the last drop out of the tip which is held against the Bide of the receiving vessel. 

e. Capillary pipets (Fig, 11, p. 14a) are usually calibrated "to contain" and 
should be rinsed out thoroughly 6-8 times with the diluting fluid used In the particular 
determination being carried out. 



14 



Transfer 


Measuring 


Serological 


Ostwald-Folia 


Capillary 


Folin-Wu 


pipet 


P*et 


pipet 


pipet 


pipet 


pipet 



Buret 










TD 
25rrt 
20°C 



\y 



i 



=_i 



=j 



3-> 



sJ 



=JB 



P 



b 

■i 



i_i 



* : 



a : 



3^_= 



UJ 
fl 



/ \ 



a mi 

zo*c 






w 



V 



of 



TO 



A 




Figure 7 



Figure 8 



Figure 9 



Figure 10 



Figure 11 Figure 12 



Figure 13 



f, " Folln-Wu " plpets (Ftg, la, p. Ha) arc a special type of measuring pipet de- 
signed to measure the fluids Involved in the preparation of the tungstlc acid protein 
free filtrate (p. 70). It Is designed to drain to the tip and Is not a "blow-out" tgog. 

4. Burets (Fig. 13, p 14a) are graduated tubcB with a uniform bore which are used 
for measuring the volume of solutions In titrations. They are available in many sizes, 
of which perhaps the 50 ml. capacity is the most commonly used. Micro-bureta are 
those with a total volume of 5 or 10 ml. down to those of 0. 1 ml. total volume. 

They should be thoroughly clean before use so that a uniform film of liquid 
remains on the walls when the liquid Is allowed to flow out. Care should be exereised 
In reading a buret to place the eye on a level with the lower part of the meniscus of 
the solution, so that parallax will not introduce serious errors. In measuring wfth 
the buret always allow one minute to elapse before taking a reading. This allows 
time for drainage from the walls of the buret. 

Outlet valves to control the flow of liquid from burets are In general of two 
types: (a) a ground-glasB stopcock which gives excellent control and can be used with 
most solutions although strong alkali solutions tend to etch and "freeze" the stopcock 
if they are allowed to remain for long periods; (b) a rubber tubing connection between 
a glass tip and the end of the buret is the main second type. The control is effected 
either by a pinch-clamp or by a glass bead which obstructs the lumen of the rubber 
tubing and is controlled by a pinching of the rubber to one side of the glass bead, to 
produce a slight crease through which liquid can flow. 

Glass stopcocks require some care in lubrication in order that they may give 
good service and still avoid undue plugging of the bores. A minimum of lubricant 
should be used. The stopcock with its adjoining connections must be absolutely clean 
before it is lubricated. Commercial pipe cleaners are useful In cleaning connections 
and bores. Old grease may be removed by scrubbing with a little ether. Lubricant 
which enters the bore of the stopcock must be removed. Such entrance may be mini- 
mized by applying only a minimal amount of grease In a ring about both ends of the 
stopcock and working this in by turning the core back and forth in its casing without 
complete rotation. A well lubricated stopcock should appear transparent throughout 
and should turn with perfect ease. Excessive pressure should not be exerted on the 
cock during lubrication or in the subsequent use of the apparatus. Such pressure tends 
to drive out the lubricant and to wear out the cock. If bits of lint or other foreign 
materials are left between the core and casing, they will etch the cock during lubrica- 
tion and cause channels with resultant leakage. 

Micro-burets are of many different types. The syringe screw burets are very 
convenient and many use a micrometer caliper with a plunger to force and to measure 
the delivery of the fluid. The micrometer scale is then used in reading. 



15 



Notes : 

"Blow-out" type pipets commonly are Identified by an etched or sandblasted ring 
near the upper end. 

One should always Inspect the markings on a buret or plpet carefully to determine 
the capacity, the type, and especially the calibration Intervals. Both burets and pipets 
are calibrated to deliver water, and will not deliver with equal accuracy fluids that dif- 
fer greatly from water In viscosity, cohesion or surface tension. When water Is run 
out of a buret or plpet, a film of liquid remains adherent to the walls. The more rapid 
the rate at which the vessel Is emptied, and the greater the viscosity of the fluid, the 
thicker this film will be. In order to deliver amounts constant even to within one part 
per hundred, It is necessary that the surface of the liquid descend in the vessel at a 
sufficiently slow and steady rate. In this wny the residual film will be thin and constant. 

Precautions In the Use of Pipets : 

1. Never insert a plpet into the stock reagent bottle. If the plpet is dirty or some 
untoward Incident occurs, the entire stock of reagent may be ruined. Instead, pour 
some solution out Into another container (a beaker or test tube) and pipet from this. 
Discard the excess reagent — never return the excess to the stock bottle. 

2. The plpet must be clean and show no droplets on the wall after draining. 

3. Before use the pipet must be dry or rinsed three times with the solution it is to 
measure. 

4. Do not pipet poisonous or corrosive reagents without safety devices Buch as a 
long rubber tube between mouth and pipet. 

5. Fill the pipet to above the mark with the solution to be transferred, using gentle 
suction with the mouth and then close the upper end of the tube with the dry index finger. 
The tip of the plpet Is then wiped off to remove any adhering droplets, and the solution 
is then allowed to flow out slowly with the plpet held In the vertical position, until the 
menlscuB coincides with the mark, and the tip is then "touched off" to the side of the 
vessel. The pipet Is carefully moved to the delivery vessel and the fluid allowed to de- 
liver freely with the tip in contact with the inclined wall of the vessel. After free out- 
flow has ceased, keep the tip in contact with the wall for about fifteen seconds. 

6. If the pipet is the blowout type expel the fluid in the tip as outlined above under 
the Ostwald-Folln pipet. If It is the "to contain" type it must be rinsed out as outlined 
under that type. 



< 



16 



I 



Volumetric analysis - goivcru] pri nciples : 

In volumetric ;uialysla, one solution (generally In a buret) Is added to a carefully 
measured volume of another reagent, in a beaker or flask. The addition from the buret 
is continued until the reaction Is seen to be complete. This Is known as the "stoic ido- 
metric end-point" or merely as the "end-point" of the titration. It is usually indicated 
to the analyst by a rapid c lunge of some sort pecurring in the solution such as a change 
of color, turbidity, etc. Very often, the change is due to a change in an auxiliary re- 
agent known as an "indicator" which has been added for the purpose. In other cases the 
end point may be indicated by the appearance of an excess of the titrating substance in 
the solution being titrated. The titrant itself is thus serving as its own Indicator. 

In acid-base titrations the indicators are weak acids or bases which have a different 
color in the form of their salt. In oxidation-reduction indicators, the oxidized form of 
the indicator has a different color from that of the reduced form. The choice of an Indi- 
cator will depend upon the particular reaction being carried out and it should indicate 
the point of chemical equivalence of the two reactants - that is the "stoichiometric end- 
point." 

For acid-base reactions it may be said that, in general, the Indicator should show 
its color change at an acidity (or alkalinity) corresponding to that of a solution of the 
pure salt formed In the titration. For example: the pH or -Jog(lv ), (which is a measure 
of the acidity of the solution) (see page 40) of a 0. 05 N solution of sodium acetate which 
is the product of the reaction of 0. 1 N NaOH and 0.1 N CH 3 COOH (acetic acid) is about 
8.8, slightly on the alkaline side of acid-base neutrality (pH7). Therefore, to attain 
stoichiometric or chemical equivalence at the end-point, we should choose an indicator 
such as phenolphthaleln which changes color close to 8. 8 (instead of one changing close 
to pH7). 

In the case of the titration of 0.1 N HC1 with 0.1 N NH 4 OH, the solution at the stoich- 
iometric end-point will be 0. 05 N NH 4 Cl with a pH of about 4.6; the Indicator of choice 
here is one that changes color on the acid side of acid-base neutrality close to 4. 6 so as 
to achieve stoichiometric equivalence at the end-point. Methyl orange or methyl red are 
such indicators. The choice of an indicator is not always a simple matter. It is best 
to use the indicator recommended in the procedure used, if at all possible. 

Volumetric standards, like volumetric measuring apparatus, can be accurate to one 
part in 1000. For convenience in computation, concentrations of solutions are expressed 
in terms of normality, i.e., 2 N, 0.5 N, or 0.01 N. The capital N, underlined, Is the 
usual abbreviation for normal. For further definitions see p. 11. 

A given number of ml. of any solution is equivalent to the same number of ml. of 
any other solution of the same normality if it Is used for the same type of reaction. 
Thus 1 ml. of 0. 5 N HC1 Is equivalent to 1 ml. of 0. 5 N KOH and equivalent to 1 ml. 
0. 5 N H 2 S0 4 because each Is equivalent to the same amount of hydrogen. If one has a 

17 



solution of either acid or alkali whose normality Is known, the strength of other acids 
and alkalies can bo determined with ease. 

Since the molecular weight of 1IC1 is 36.46 and of NaOil Is 40. 00, It follows that 
36.46 g. of 1IC1 contain the same number of molecules as 40.00 g. of NaOH. If 36.46 g. 
HC1 and 40.00 g. of NaOH are each dissolved In water and diluted to exactly one liter, 
each liter will contain the same number of dissolved molecules, and each ml, will con- 
tain the same number of molecules. The two solutions are equivalent to each other and 
are each 1 (one) normal In concentration. 

The HC1 is 1 (one) normal because it contains 1 g. (1.0080 g.) of replaceable 
hydrogen in one liter of solution, and the NaOH Is 1 (one) normal because it contains 
17 g. (precisely 17.0080 g.) of hydroxyl ion (OH") which is equivalent to 1 g. of hydrogen. 
The molecular weight of H 2 S0 4 is 98. A solution containing 98 g. of HgSO, per liter con- 
tains the same number of molecules per unit volume as the HC1 solution of 36.46 g. HC1 
per liter. But each molecule of H 2 S0 4 contains 2 atoms of replaceable hydrogen. The 
solutions are not equivalent since the HgSO. contains 2 g. of replaceable hydrogen per 
liter; it is twice as strong as the HC1 or 2 x N. To make a normal solution of HgSO., 
take 98/2 = 49 g. H 2 SO^ per liter of solution. Thus a normal solution contains one 
equivalent weight of the substance in a liter of solution. Then one milliliter of a normal 
solution will contain one milllequivalent weight of the substance , or the same number of 
milligrams of the substance as one liter contains grams of it. Hence 1 ml. of a 1 N solu- 
tion of HCI contains 36.46 mg. of HCl; 1 ml. of 1 N NaOH contains 40 mg. of NaOH; 
1 ml. of 1 N H„S0 4 contains 49 mg. of H„S0 4 . The number of milliequivalents (abbre- 
viated meq. or mEq.)of a substance present in a given volume of a 1 N solutioals equal 
to the number of ml. If the solution is 0,6 N, there is in each ml. 0.5 meq. and in 10 ml. 
there would be 10 x 0. 5 = '5 meq. 

It Is always true that the product of the number of ml. times the number of milli- 
equivalents in each ml. (the normality) gives the total number of meq. present. Since 
dilution does not change the amount of solute present, the volume of the original solution 
(Vj) times ltB normality (N^) equals the volume of the diluted solution (V" 2 ) times the 
new normality (N 2 ) , or 

N 1 V 1 s N 2^2 - meq P re8ent 

If three of these quantities are known, obviously the fourth can be calculated. 

If It is established by titration that a given volume of an unknown solution (Vj) , re- 
quires a determined volume (V" 2 ) of a solution of known normaUty (N 2 ) to reach an equi- 
valence point (stoichiometric end-point) , the same equation can be used to calculate the 
normality of the unknown solution, N.. 



18 









GENERAL INSTRUCTIONS FOU THE PREPARATION OF SOLUTIONS 

Certain standard solutions can be directly and accurately prepared by weighing out 
a calculated amount and dissolving up to a predetermined volume. These are known as 
primary standards. Others arc standardized directly or indirectly against those which 
have been prepared by weight directly. All solutions are so prepared that a certain 
quantity of solute is contained in a known volume. In general, volumetric standards, 
like volumetric measuring apparatus, should be accurate to one part In a thousand* 
Normal and fractional normal solutions are not always stable and must be rechecked 
if they have been standing some time, These Instabilities will be noted under each sol- 
ution as it is mentioned. 

In many Instances, solutions are required, whose concentration need be known less 
accurately or in which small variations are not important to the results of the analysis. 
In these the concentration may be expressed less precisely, commonly as a percentage. 
By this (for our use) is meant grams of substance per 100 ml. of solution and is some- 
times expressed as " W/Y" or "weight per volume. " Where fluids are dissolved In fluids 
the short-hand form "V/V" may be used. 

The attempt to be concise In the description should not lead to ambiguity. Such 
expressions as "dilute one to three with water" are not precise and lead to mistakes. 
Also when the article of commerce Is not a pure substance but a solution in water such 
as concentrated HC1 (about 37% HC1 by weight) a description of a solution as a 10% solu- 
tion of HCl does not allow the reader to choose with any degree of certainty between 
3.7% by weight of HCl and 10% by weight HCl. 

Accurate solution preparation is essential to good analytical work. However, the 
degree of accuracy required varies from one solution to another. Primary standards 
as discussed below should be prepared with maximum volumetric accuracy, I.e. , to 
about 0. 1%. Other solutions, because of the less critical part they play in the reaction 
may need to be accurate only to 1% or so. In the description of solutions in each method 
an indication of the degree of accuracy required is shown by the number of significant 
figures included. 

Thus, the weight of a primary standard required will be stated to tenths of a milli- 
gram (as 4.6827 g.) while the weight of ammonium oxalate required In a saturated solu- 
tion will be stated to only two significant figures (as 15 g. ). 

Technique of solution preparation : 

In general, the technique of the preparation of solutions is to measure as accurately 



19 



as necessary by weighing, pipetting or by using a graduated cylinder, etc, , the required 
amount of solid or liquid and then by adding water or other fluids to bring to a specified 
volume. Only In the case of the preparation of primary standards or of dilution of accur- 
ately standardized solutions or in the dilutions of unknown solutions, is It necessary to 
use volumetric flasks. Their use should be avoided where their accuracy Is not needed 
because they are both fragile and costly. Solutions prepared in volumetric flasks should 
be transferred Immediately to other containers for storage. 

When diluting acids and bases or In any case where heat Is generated by dilution, 
ALWAYS POUR ACID (OR ALKALI) INTO WATER, to avoid local boiling and spattering 
of the solution. All solutions should be adequately mixed to homogeneity. A good rule 
In using volumetric flasks is to invert, shake well and then bring to an upright position 
again, allowing time for the air bubble to travel to the top each time. This should be re- 
peated at least ten times after all solid material has disappeared from the solution. 

PRIMARY STANDARDS 

All volumetric and indeed all quantitative determinations depend on the availability 
of pure compounds and the ability to prepare accurate stable solutions. The pure sub- 
stances which are best for this purpose are called "primary standards." A primary 
stands rd should have the following characteristics. 

1. Stable and definite composition. 

2. Stable to drying without decomposition. 

3. Large equivalent weight (to minimize the weighing error). 

4. Analyzable with accuracy. 

5. The reaction in which it Is Involved as a standard should be 

a. Single 

b. Well defined 

c. Rapid 

d. Complete 

Some of the presently available primary standards, their preparation, and some of 
their uses are listed below. 

Primary Standards : 

Acids : 

1. Constant Boiling HC1 M.W. 36.46 

Ref: Hylett, G.A. , and W.D. Bonner. J. Am. Chem. Soc. , 31, 390 
(1909). 
Standard hydrochloric acid is easily prepared by first preparing diluted HCl by 
adding about 650 ml. of distilled water to about 1000 ml. of concentrated HCl (analytical 



20 



t 



grade). Tliis mixture is then slowly distilled In an all-glass Btlll at the rate of 3-4 ml. 
per minute. After about 3/4 of the mixture has been distilled, the rest of the distillate 
la collected until not Icbs than 50-GG ml. remain In the flask. This distilled fraction 
la constant in composition (that is, It does not change with time) and Is not noticeably 
hygroscopic or volatile. The exact composition of the distilled fraction depends only 
upon the barometric pressure at the time of distillation, according to the following table. 



Barometric % HC1 by Weight of HC1 solution 
Pressure weight needed for one mole of 
HC1 (grama) * 

770 20.197 180.407 

760 20.221 180.193 

750 20.245 179.979 

740 20.269 179.766 

730 20.293 179.555 



By the use of a small plpet, it is a simple matter to weigh out the required amount of 
acid to less than 10 mg. and thia is sufficiently precise to provide acid more accurate 
than that attained by most other procedures. The weighing of this acid In an analytical 
balance is permissible since it is non-volatile, but care should be taken to avoid spill- 
ing of even small droplets since the liquid Is, of course, corrosive. 

2. Potassium Acid Phthalate - KHCqH^ M.W. 204.22 

This can be obtained in very pure form (99. 95%) and 1b highly recommended as 
a primary standard. This salt crystallizes without water of hydration, It is not hygro- 
scopic, and may be dried at 110-115°C. without any danger of decomposition. It is ob- 
tained from many different manufacturers and is available from the National Bureau of 
Standards for a reasonable fee. The latter supplies two types, one especially for tltrl- 
metric standardization, and the other for a pH standard (see below>. 






0.1N KHCqH.O. — Weigh out 10. 207 g. of dry potassium acid phthalate on an analytical 
balance and dissolve the crystals In about 200 ml, of distilled water. Transfer quanti- 
tatively to a 500 ml. glass-stoppered volumetric flask, add distilled water to the mark 
and mix. This solution should be kept In a glass stoppered Pyrex bottle and stored in 
the refrigerator when not being used. It may be used to standardize alkali (except am- 
monia and other weak bases) using phenolphthaleln as an indicator. This solution should 
be reasonably constant for many months If handled with ordinary care. 



21 



3. Potassium bl-lodate - KH(IO ) M.W. 389.94 

This salt Is a strong acid ana can be used with any Indicator with a color change 
between pH 4 and 10, It Is anhydrous, non-hygroscopic and thus can easily be kept 
without change on the shelf. 

Standardization of Alkali: 



Procedure : 

1. Using standard acid solutions : Into a 250 ml. Erlenmeyer flask, plpet accurate- 
ly 25.00 ml., of the standard HCl solution. Add two to four drops of 1% alcoholic phenol- 
phthaleln indicator solution and add the alkali (e. g. , NaOH solution) from a buret to a 
permanent light pink color using care at the end-point. The same technique can be used 
for standardization by potassium acid phthalate but weak bases (e. g. , ammonia) should 
not be used. 

2. Using solid standards : . 8 to 0. 9 g. samples of KHCgf^C^ or 1. 6 to 1. 7 g. 
samples of KH(I0 3 ) 2 are weighed out in beakers or flasks very accurately using an 
analytical balance. The salt is dissolved In about 25 ml. of water and titrated with the 
base to be standardized using phenolphthalein as the indicator. The normality of the 
base Is calculated as Illustrated In VOLUMETRIC CALCULATIONS page 32 , using 
the molecular weights given above and assuming the salts act as acids with one re- 
placeable hydrogen per molecule. 

Bases : 

1. Sodium tetra-borate (borax) Na^cy 10H 2 O M.W. 381.43 

Sodium tetraborate decahydrate is obtained in a pure condition by two recrystal- 
llzations of a good product from water, followed by drying to constant weight In a desic- 
cator over a solution saturated with respect to both sucrose and sodium chloride. To 
expedite drying, washing with 95% alcohol and with ether, as recommended by Hurley, 
F.H. , Anal. Chem. 8, 220 (1936) and ibid. , 9, 237 (1937) may be resorted to. In tight- 
ly closed containers the decahydrate seems to have considerable stability up to at least 
12 months. 

2. Trls (hydroxymethyl) amino methane H 2 NC (CH„OH) 3 M.W. 121.14 
This organic base can be regarded as a monovalent base and has a number of 

Important advantages. 

(1) Relatively high equivalent weight 

(2) Low moisture absorption - about that of potassium acid phthalate - 
requires no further drying 

(3) CO„ absorption is absent 

(4) Long shelf-life stability 

(5) Reacts stoichiometric ally 



Oxidation-reduction: 

1. Potassium iodate (KIQ 3 ) M.W. 214.02 

Potassium iodate, can be secured in an extremely pure form and used as a 
primary standard. The salt should be dried to constant weight In a desiccator or In a 
drying oven (106°C). Each molecule of this salt, when reacting with an excess of 
potassium Iodide in the presence of acid, liberates 6 atoms of Iodine. 

KI0 3 / 6 KI / 8 HC1 = 6 KC1 / 3 H 2 / 8 I (or 3 Ij) 

Therefore a Uter of 0. 1 Nl iodate contains 1/60 of a gram-molecular weight. 0. IN KIO. 
is prepared by weighing out 3. 5670 g. of KlOg on the analytical balance. Dissolve the 
salt In 200 ml. of distilled water, transfer quantitatively to a one liter volumetric flask 
and dilute with distilled water to the mark. This solution should be stored In a glass- 
stoppered bottle in a cool place. It should keep Indefinitely. 

2. Potassium bl-iodate KH(I0 3 ) 2 M.W. 389.94 

This salt, which can be regarded as an equi -molecular mixture of KlOg and HlOn 
can be obtained pure and Is non-hygroscopic even in the anhydrous state. The reaction 
is similar to that given above for iodate but one molecule of the bi-lodate liberates 12 
atoms of iodine bo that the preparation of a liter of 0. 1 N KH(I0 3 ) 2 (for this reaction) 
requires only 3, 2496 g. Note that above under acid primary standards to prepare a 
0. 1 N_ solution to be used in acidimetry would require 38. 9940 g. per liter. 

3. Sodium oxalate (Sorenson salt) (Na 2 C 2 04) M.W. 134. 01 

This salt can be obtained in high purity. It is recommended over oxalic acid for 
the standardization of permanganate because when prepared as indicated it forms a very 
stable solution. When exposed to light a slow decomposition may take place yielding 
carbon dioxide, carbon monoxide and water. 

0. 1 N sodium oxalate 1b prepared as follows: Dry about 10 g. of the pure salt 
in a drying oven at 150 °C. for 3 to 4 hours. Cool in a desiccator. Weigh out 6. 7010 g. 
(M.W, /20) on an analytical balance, dissolve In distilled water and transfer quantita- 
tively to a liter volumetric flask. Add 30 ml. of concentrated sulfuric acid (analytical 
reagent). Cool, dilute to the mark with distilled water and mix thoroughly, It should 
keep at least a year. The sulfuric acid is essential to long-term stability. 

Argentimetric and Mercurlmetrlc . 

1. Silver Nitrate (AgNO^ M. W. 169. 89 

Very pure preparations of silver nitrate can be obtained. Working In subdued 
light, the crystals should be pulverized and then dried for 3 to 4 hours at 150 C. They 
should be stored in a desiccator protected from light. 

To prepare bO.IN solution of AgNO„ proceed as follows: Weigh out exactly 
B. 4945 g. of the dried salt, transfer to a 600 ml. volumetric flask and dilute to the 
mark with distilled water. Mix. Keep in a brown bottle la the dark, 

23 



2. Sodium Chloride NaCl M.W. 58.45 ( 

It la convenient to have available a standard chloride solution to check the silver 
nitrate (or mercuric nitrate) solutions after a lapse of time. For our purposes the ana- 
lytical reagent grade sodium chloride Is sufficiently pure. 0. 1 N NaCl can be prepared 
as follows: 2. 9225 g. of the salt which has been dried at 150°C. for 12-14 hours Is 
weighed out on an analytical balance, transferred quantitatively to a 500 ml. volumetric 
flask and dissolved and diluted to the mark with distilled water. Mix well and store pro- 
tected from the light. 

3. Potassium thlocyanate KSCN M.W. 97.18 

Prepare the anhydrous salt by re crystallizing from water one to three times, dry 
over PqOe In a desiccator for three days, heat at 150°C. for one hour, melt at 200°C. 
for 10-20 minutes, cool, grind to a powder and store over PgOg, for a few days. There- 
after, It Is* stable In atmospheres less than 45% relative humidity. For a 0. 1 N solution 
dilute 4.8590 g. to 600 ml. in a volumetric flask. 

pH Standards: for the glass electrode 

ft is always best to use a pH standard which is close to that of the unknown. It Is also 
best to use two different pH standards so as to check the response slope of the Instrument, 
and thus to insure the correct operation of the instrument before readings are made. 
See below under pH meter, glass electrode p. 46. 

Acid Standards ; 

1. Potassium acid phthalate 0.05 MpH = 4.01 £ 0.01at25°C. 

Weigh out on an analytical balance 10. 211 g. of pure dry salt, dissolve up to 1 
liter In a volumetric flask. Alternatively, It may be prepared from a stock 0. 1 H solu- 
tion (see acid primary standards). This dilute solution is good for a few days and then 
should be discarded. 

2. Potassium acid tartrate 0.034jtf pH = 3.58^ 0.01at25°C. 

An aqueous solution saturated at between 22 - 28°C. An error of 10% in con- 
centration produces the given pH with an error of about 0, 01 units. 

3. Potassium tetraoxalate 0.05 M KH 3 (C 2 4 ) 2 - 2H 2 pH ; 1,68 i 0.01at25°C. 

A solution of this salt re crystallized from water Is pure enough for the prepar- 
ation of the standard. Dissolve 12. 7095 g. and dilute to 1 liter with water. 



24 



( 



4. Hydrochloric acid 0.1 N pH « 1.10 4 0.02at25°C. 

Prepare exactly 0.1N HCl as Indicated under secondary standards p. 28. 

Al kaline Standards 

Standards for pH values higher than 7 are complicated by the absorption of CO„ from 
the atmosphere and the presence of CO„ In the water used In the preparation. Freshly 
boiled water should be used and the standards should be protected from the atmosphere 
as much as possible. 

1, Sodium tetraborate decahydrate 0.01 N Na 2 B 4 7 - 10 HgO 

pH s 9.18 t 0.01at2B°O. 

Dissolve 3. 8143 g. of the pure salt in 1 liter freshly boiled, cooled distilled 
water. The presence of slight amounts of the pentahydrate will not affect the final pH to 
any significant degree. This is not of course true when the acidlmetrlc standard is 
being prepared. 

2. Sodium hydroxide 0.1 N NaOH pH - 12.88 t 0.03 at 25°C. 

Prepare as described under stock reagents below, (seep. 26) and standardize and 
adjust to exactly 0. 1 N. 






( 















SECONDARY STANDARDS 



Certain substances, although relatively stable once a solution has been prepared, 
do not have the characteristics required of a primary standard, (see p. 20). They are 
prepared In approximately the correct concentration, and then their exact concentra- 
tion Is determined by titration against the appropriate primary standard. 

Although the method of standardization of working secondary standards Is usually 
given in detail under each particular method, a recommended procedure will be outlined 
here for some, since these solutions are of general application in volumetric work. 

Stock solutions: 



In the following list are Included the usually available acids and alkalies giving the 
data required for the preparation of more dilute solutions. 






Name 
%<W/W) 
Formula 



Specific 
gravity 



Molcular 
Weight 



N of cone, 
reagent 



Ml. cone, reagent 
needed for 1 liter 
1 M solution 



Acetic acid 


1.049 


99. 6% CHgCOOH 




Hydrochloric acid 


1.19 


36.0% HC1 




Nitric acid 


1.42 


69.5% HNO 
Phosphoric acid 




1.71 


85.0% H 3 P0 4 




Sulfuric acid 


1.84 


96. 0% H 2 S0 4 




Sodium hydroxide 


1.51 


48% NaOH 




Ammonium hydroxide 


0.89 


68. 6% NH.OH 





60.05 


17.4 




67 


36.47 


11.6 




86 


63.02 


15.4 




65 


98.00 


14.6 M 




68 (1M) 


98.08 


IT. 8 M 




26 


40.01 


18* (see 


below) 


69* 


35.05 


14.8 




67 



♦The concentrated NaOH is described in the list of stock reagents below. 

The normalities and volumes mentioned above are approximations only, with 
enough allowance for variations so that the solutions when prepared will have a con- 
centration greater than 1 N (or 1 M in the case of HgPO^). 

Carbonate -free concentrated NaOH: 

Solid sodium hydroxide always contains some carbonate and readily takes up 
moisture and carbon dioxide from the air, A standard solution of this alkali cannot be 
made by dissolving a calculated quantity in the required amount of water. Fortunately, 



27 



sodium carbonate 1b practically insoluble In a saturated solution of sodium hydroxide. 
Therefore, a stock saturated solution of the alkali should always be kept on hand for 
the preparation of standard solutions, 

Weigh hastily in a Fy rex beaker on a solution balance, 1100 g. (approximately) of 
the best quality {analytical grade) sodium hydroxide and place It in a two liter Pyrex 
beaker or flask. Add immediately one liter of distilled water and stir continuously until 
all the solid la dissolved. The solution becomes very hot. Allow to cool. After cooling, 
store in a Pyrex bottle, stoppered with a rubber stopper. Standardization of this con- 
centrated solution is not advisable. It can be considered to be about 18 N in making up 
dilutions. 

. . .1 N Sodium hydroxide NaQH : 

For each liter of , 1 N NaOH to be made, use 5. 9 ml. of the clear concentrated 
saturated NaOH (18 N). Dilute with freshly boiled and cooled diBtilled water to the re- 1 
quired volume. 

Standardization : Titrate with 0. 1 N HC1 whose normality is accurately known, using 
phenolphthalein as the indicator. Do not shake more than necessary to mix contents 
during the titrating. The solution may also be standardized against standard acid phtha- 
late or acid lodate solutions or weighed out samples (Bee p. 22) with phenolphthalein as 
the indicator. In all acid-alkali titrations the alkali is placed in the buret, to avoid 
absorption of CO„ from the air, 

0.1 N Hydrochloric Acid HC1 : 

Measure about 17.5 ml. of the concentrated reagent grade hydrochloric acid (36% 
by weight) in a cylinder and pour the acid into a two liter volumetric flask, dilute to Ahe 
mark and mix well. Determine the exact normality by titration against standard 0. 1 
N NaOH solution or by the use of sodium tetraborate (borax) either as a solution or as 
weighed-out samples. Tria (hydroxymethyl) amino methane may also be used as the 
primary standard. 

0.1 N Potassium permanganate KMnO ^: 

Although the pure crystals of potassium permanganate can be weighed accurately, 
it is not practical to use this salt as a primary standard because the solution tends to 
become weaker on standing. Avery low concentration of organic substances In the dis- 
tilled water used for the preparation of the solution slowly reduces the permanganate. 
The potassium permanganate solution is therefore prepared slightly stronger than is 
desired and allowed to Btand undisturbed and tightly stoppered for a weekbefore it Is 
standardized with a sodium oxalate solution. In most cases it is advisable to standard- 
ize ,the permanganate each day before use. 



28 



( 






Dissolve about 3. 5 g. of KMn0 4 (analytical reagent) In about 1100 ml. of distilled 
water. Set aside In a tightly stoppered bottle for about one week. During this period 
the solution should not be disturbed so that any small quantity of manganese dioxide 
formed may settle to the bottom of the bottle. Carefully siphon off the clear superna- 
tant liquid Into a dark brown bottle. 

Standardization ; Accurately pipet 20. 00 ml. of 0. 1 N sodium oxalate primary 
standard Into a 500 ml. Erlenmeyer flask. Warm the container to 70-75°C. and titrate 
with the potassium permanganate solution until a faint pink color, that is given by the 
addition of a single drop, persists for one minute. If the titration Is carried out slow- 
ly, the solution will become cool and the reaction slowed up. The solution in the flask 
■ hould be kept at about 70°C. throughout the titration. The first few drops are decolo- 
lsed very slowly but later the permanganate may be added rapidly. A check titration 
should agree with the first within 0.1 ml. Many analysts consider It unnecessary to 
adjust the strength of the permanganate solution to exactly 0.1 N, since a simple cor- 
rection can be made in the calculations by using a "factor." See below. The perman- 
ganate solution should be kept In a dark brown bottle or one covered to keep out light. 
The titer of the solution may change somewhat, immediately after it has been freshly 
prepared but after standing for a week or so the strength is generally constant. 

I.1H Sodium Thiosulfate Na. B.O, : 
— — — a % 3 

Weigh out about 25 g. reagent grade sodium thiosulfate fjNhgSwpg'5 HgG) and 5 g. 
of sodium tetraborate (borax) (NagB^Of- 10 HgO). Transfer to a Alter flank and dis- 
solve In about 800 ml. freshly boiled and cooled distilled water. Add about 7 ml. of 
Iso-amyi alcohol and shake to dissolve. Dilute to about one liter. Transfer to a pyrex 
bottle and store for a few days before using. 

Sodium thiosulfate solutions deteriorate on standing. Ibis is stated to be doe to 
decomposition of the salt by traces of CO„ in the water, to bacterial decomposition, 
and to slow oxidation by dissolved oxygen. The purity and crystal water content are not 
sufficiently constant in the reagent grade to allow its use as a primary standard. The 
solution should always be made with boiled distilled water; the solution should be made 
alkaline and allowed to stand several days before standardization. The Iso-amyl alcohol 
Is added to minimize bacterial action. 

Standardization: The thiosulfate is usually standardized by and used in reactions 
of iodimetry. The thiosulfate reacts with iodine as follows: 

2 Na 2 S 2 3 / Ig - 2 Nal / Na 2 S 4 6 (sodium tetrathionate) 

Thiosulfate solutions are best standardized with KK> 3 (potassium lodate) or 
KIO3- HIO3 (potassium bl -lodate) prepared as 0. 1 N solutions. With a plpet measure 
25.00 ml. of 0, 1 N lodate or bl-iodate solution Into a 260 ml. Erlenmeyer flask. Add 

29 



about 10 ml, of a freshly made 10% solution of KI (potassium Iodide) and about 20 ml. of 
I 1 N hydrochloric acid (HCl). Iodine Is liberated in this reaction thus; 

KI0 3 / 5 KI / 6 HCl = 3 I 2 / 3 HgO / 6 KC1 

Titrate the liberated iodine with thlosulfate from a 50 ml. buret. The thiosulfate is de- 
livered into the iodate -potassium iodide-acid solution until the iodine color has faded to 
a very pale yellow. One nil. of starch test solution (see below) is added and the titration 
continued until the disappearance of the blue color. 

Standard thiosulfate solutions weaker than 0, 1 N are best made by diluting the 0. 1 N 
standard with freshly boiled and cooled distilled water with an appropriate pipet and 
volumetric flask. If kept over a day these solutions should be checked with a diluted 
standard made from the potassium Iodate primary standard. Store protected from the 
COg of the air. 

2/3 N Sulfuric Acid H 2 S0 4 

Weigh out 35 g. of concentrated sulfuric acid in a small tared beaker, dilute to 1 
liter with distilled water and mix. Check by titration against standard NaOH using phen- 
olphthalein as an indicator and adjust to 0. 667 N. 

1/12 N Sulfuric Acid H 2 S0 4 

Add 2, 5 ml. of concentrated sulfuric acid to 1 liter of distilled water, Mix well and 
check by titration against 0.1N NaOH so that 20 ml. of the acid requires 16. 7 ml. of 
0. 1 N NaOH for neutralization using phenolphthalein. 

2-5 g Sodium Hydroxide NaOH 

Dilute 150 ml, of stock concentrated NaOH up to 1 liter with distilled water. Titrate 
against standard 0. 1 N HCl and adjust to exactly 2. 5 N. 

The Use of a "Factor" : 

The calculations and adjustments of concentration are made using the usual formula; 
V 1 N 1 - V 2 N 2* ^ many cases, adjustment to exactly the nominal normality is not nec- 
essary. In this case, the use of a factor is convenient. The factor for any given solu- 
tion is defined as follows: 

_ . Actual Normality 

Nominal Normality 

Example: A solution of NaOH is made up to ue close to 0. 1 N. Titration shows it to be 
0. 0987 N. The factor is then 0. 9870. If the titration had shown it to be 0. 1032 N the 
factor would have been 1.032. 

30 



( 



( 






S tarch Test Solutions ; 

1. Triturate (grind) 1 g. of arrowroot starch with 10 ml. of cool distilled water 
and pour slowly with constant stirring Into 200 ml. of boiling distilled water, Boll the 
mixture until a thin, translucent fluid la obtained. Allow to settle and use only the clear, 
supernatant liquid. Longer boiling than necessary renders, the test solution less sensi- 
tive. The solution must be freshly prepared. 

2. Another popular method tor making a solution of starch Is to suspend 100 g. of 
pure cornstarch (Klngsford's) In approximately one liter of 0.01 N hydrochloric acid 
with shaking at frequent intervals for one hour. Decant the supernatant liquid after sed- 
imentation. Wash twice, with one liter each time, with 0.05 per cent sodium chloride 
solution. Spread out and allow to dry In the air. Thoroughly grind 16 g. of this washed 
starch in a mortar with 50 ml. of distilled water and pour Into BOO ml. of boiling dis- 
tilled water. Boll for 1/2 to 1 minute without agitation. Cover the mouth of the flask 
with a beaker and set In a boiling water bath for 15 to 30 minutes. A few drops of this 
solution are sufficient for a titration. 

3. A one per cent solution of a high grade soluble starch (Merck's soluble starch) in 
cold distilled water has been also successfully employed for lodometric titrations. 
Most starch solutions deteriorate quickly on standing with the growth of molds. The solu- 
tion of Plncussen, which is made by dissolving 1 g. of soluble starch in 10 ml. of boiling 
distilled water and adding 90 ml. of saturated sodium chloride solution, keeps satisfac- 
torily. 
























31 



VOLUMETRIC CALCULATIONS 



A. Solid Standard Standardization of NaOH 

Samples of potassium acid phthalate (KHCgH.O.) M.W. 204.22, are weighed out 
and dissolved In about 25 ml. of distilled water and titrated by the unknown sodium 
hydroxide solution, using phenolphthalein as an Indicator, Three samples gave the 
following results: 

Sample Weight (g.) ml, NaOH * meg, total meg, /ml. NaOH-Ofl (calc. ) 

1 0.8436 41.24 * 4.131 0.1002 

* 

2 0.8978 43.91 * 4.396 0.1001 

* 

3 0.8621 42.05 * 4.221 0.1004 

* 

1. The weight of the samples divided by the molecular weight of potassium acid 
phthalate multiplied by 1000 gives the total meq. of acid (and of alkali). 

2. Since this number of milllequivalents is present in the indicated volumes of 
NaOH, division of total meq. present by number of milliliters of NaOH gives meq. /ml. 
or normality of the NaOH. 

B. To prepare a Normal Solution from a Concentrated Acid Solution : 

1. Find the normality of the concentrated acid. 

a. Multiply the specific gravity by the assay (% by weight) to get g. /ml. 

b. Multiply by 1000 to get g. /liter. 

c. Divide g. /liter by the equivalent weight to give meq. /liter which la by 

definition the normality of the concentrated acid. 

2. Use the formula V^Nj r V 2 N 2 

Example : Make 3 liters of 0. 16 N HNO„ from concentrated acid. 
1. Find the normality of the concentrated acid (69% by weight). 

a. 1.48 (specific gravity) x 0.69 (assay) a 1.0212 g. /ml. 

b. 1.0212 x 1000 = 1021. 2 g. /liter 

c. 1021. 2/63 (equivalent weight) - 16. 2 equivalents/liter « normality. 



32 



( 






2. 360© x 0.16 = V 2 x 16.2 

3000 x 0.16 _ 29. 6 ml. concentrated acid to be diluted to 3 liter*. 
16.2 

C. To Prepare a Normal Solution from a Percentage Solution : 

1. Find the g. /liter in a 1 N solution (one equivalent weight). 

2. Find the g. /liter in the % solution. 

3. Find the N of the % solution by division of Step 2 /Step 1. 

4. V^N, = V„N„ 

Example : Make 2 liters of 3 N HC1 from 37% HC1 (concentrated add) 

1. IN HCl = 36. 5 g. /liter. 

2. 37% HC1 has a specific gravity of 1. 19. 
1.19 x0.37 xlOOO = 440 g. /liter. 

3. 440/36.5 = 12,08 N. 

4. 2000 ml. x 3 N = V 2 x 12.08 N. 

2000 x 3 = 497 ml. concentrated HC1 (37%) to be diluted to 2000 ml. to 
12.08 obtain 3 N HC1 

D. To Prepare a Percentage Solution from a Normal Solution 

1. Determine the equivalent weight. 

2. Find the g. /liter In the percentage solution. 

3. Find the N of the percentage solution by dividing Step 2 /Step 1. 

4. VjlNj r V 2 N 2 . 

Example : Make 2 liters of 5% HC1 from 3 N HCl. 

1. 36. 5 g. /liter in 1 N HCl (one equivalent weight). 

2. 5% HCl is 5 g. /100 ml. or 50 g. /liter (W/V). 



33 






( 

3. 50/36.5 = 1.37 (normality of 5% HC1). 

4. 2000 x 1.37 = V 2 x 3N 

2000 x 1,37 _ 913 m i. 3 n H C1 to be diluted to 2 liters to obtain 5% HCl. 
3 

E. To Prepare a Percentage Solution from a Percentage Solution: 

1. Find g./ml. contained in the given solution. 

2. Find total g. needed for total amount of new solution. 

3. Then total g. needed = ml of given <j to be du u t e d to the required total volume. 

g./ml. given 






Example: Make 3 liters of 7% sulfuric acid from 15% 

1. 15% = 15 g. /100 ml. = 0.15 g./ml. 

2. 7% = 7 g. /100 ml. = 70 g. /liter = 210 g. /3 liters. 

3. 210/0. 15 = 1400 ml. of 15% to be diluted to 3 liters to obtain 7%. 
An alternative procedure is to use the relationship % 1 V 1 a %^2 

In the above example 15 x \ l - 7 x 3000 

u 7 x 3000 , J(W¥ , 

Vi = — - 1400 ml. 

15 

F. To Calculate Normalit y of a Pe rcentage Solution: 

j a 

1. Find the molecular weight of the substance, 

2. Find g. /liter of a 1 N solution. 

3. Find the percentage of a IN solution. 

4. Divide given % by 1 N %. Step 2 divided by Step 3. 

Example: Find the normality of 10% H-SCr.. 

c — j '"2 4 

1. Molecular weight H 2 S0 4 = 98 g. =2 equivalents. 
Z. IN H2S0 4 contains 49 g. /liter. 

34 









< 



( 






voltage developed is proportional to the ratios of the concentration of hydrogen tons 
inside and outside of the glass membrane. 

Commercial glass electrode pH meters read directly in pH units. They should be 
calibrated just before use by the aid of at least two standard buffer solutions differing 
by at least 2 pH units. See pH standards p. 24. 

For best results follow the instructions given by the manufacturer of the particu- 
lar instrument used. Most directions suggest using only one standard buffer. The use 
of two will detect errors due to cracked or otherwise faulty electrodes; errors not 
easily shown up by single point calibration. 



pK a Values for Some Acids Suitable for Buffers 






Name of Acid 



Formula 



PK, 



Buffers at pH 






Pyrophosphoric 

ti 

Phosphoric 
Glycine (as base) 
Phthalic 
Tartaric 
Citric 



H 4 P 2°7 



H 3 P0 4 



NH 2 CH COOH 
C 6 H 4 (COOH> 2 
HOOC(CHOH) 2 COOH 



(HOOCCH 2 ) 2 - C-OH 
COOH 






Lactic 

Barbituric 

Tartaric 

Acetic 

Citric 

Citric 

Phthalic 

Carbonic 

P J* ropho sphor ic 

Phosphoric 

Py ropho spheric 

Boric 

Glycine (as acid) 

Phosphoric 

Salicylic 



CH 3 CHOHCOOH 
NHCONHCOCH„CO 



see above 




CH„COOH 




see above 




see above 




see above 




H 2 C °3 




see above 




see above 




see above 




H 3 B0 3 




see above 




see above 




HOC 6 H 4 COOH 




47 



(1) 


0,854 


(2) 


1.959 


(1) 


1.959 




11. 647 


(1) 


2.90 


<1> 


2.96 


U> 


3.1 




3.98 




3.98 


(2) 


4.16 




4.73 


(2) 


4.75 


(3) 


5.40 


(2) 


5.51 


(1) 


6.53 


(3) 


6.54 


(2) 


6.7 


(4) 


8.44 


(3) 


9.20 




9.78 


<3> 


12.44 


(2) 


13.0 



wy 



i 

2 

2 

2.3 

3 

3 

3 

4.2 
4,2 
4.7 
4.7 
4.7 
5.4 
5.5 
6.5 
6.5 
6.7 
8.4 
9.2 
9.8 
12.4 

13. 






COLORIMETRIC TECHNIQUES 



The method of analysis known as colorlmetry has mad eselble the development of 
methods for analysis of small amounts of blood containing a*y minute amounts of ma- 
terial, This la turn has led to the present widespread knowledge of the variations In 
blood constituents In health and disease. This knowledge Is, of course, not complete 
and new methods and new applications continue to be made. Outside the scope of this 
manual Is the extensive literature of what Is called "ultra-micro analysis," since In 
most cases a few milliliters of blood will suffice using the methods In this manual, m 
pediatric work, Infants and children cannot furnish blood as easily or In such large 
amounts, so methods of analysis based on the volumes obtained by heel or finger punc- 
ture have been developed. Borne of these are Included In this manual, 

Colorlmetry depends on the quantitative comparison of the amount of color devel- 
oped In unknown solutions with the amount of color developed in solutions having a known 
amount of pure substance present. The comparison Is most often made visually, match- 
ing unknown tubes with standard tubes, or matching visual fields In a special apparatus 
such as a Duboacq colorimeter. 

This measurement technique is based on Beer's Law (sometimes called Lambert- 
Beer's or Bouger-Beer's) which states (briefly and approximately) that the amount of 
light absorbed by a colored solution is proportional to the concentration of colored ma- 
terial present. Beer's Law applies In a practical way only to solutions with a relative- 
ly small range of concentration, and only when the Incident light la "mono -chromatic;" 
(actually, a narrow band of wavelengths). These requirements are only relative and to 
some extent Interdependent. The optimal conditions for colorlmetry are best deter- 
mined Individually for each determination. 

Colorlmetrlc measuring apparatus : 

Block comparators : The block or rack comparator Is used in situations In which 
simplicity and speed (for example: bed-side analysis) require the sacrifice of accuracy. 
This method uses a series of graded standards (usually In sealed tubes) against which 
the developed color or turbidity Is compared. An example of a method In which block 
comparison 1b commonly used Is the ICTERUS INDEX, p. 187. 

Dilution Colorlmetry: This type of comparison colorlmetry uses a single, fixed 
standard and the unknown Is diluted until the standard Is matched. The Sahlt hemoglobin 
method Is an example of this type of analysis. The fixed standard In this procedure may 
be a permanent standard such as a colored glass disc. 

Comparison Colorimeter : This Is the method commonly employed for visual color- 
lmetry. The apparatus used may vary in the details but all have some arrangement 
whereby the thickness (or depth) of a layer of the standard and the unknown colored 



( 










Figure 15. Duboscq colorimeter 



48a 









solutions may both be varied and we measure the d opths of solutions that give equal 
light absorption. The Duboecq colorimeter la of this type and is quite convenient for 
this purpose (see Fig. 15, p. 48a). 

Beer's Law; 

Colorlmetry is based upon Beer's Law, which states that light In passing through 
a colored medium Is absorbed In direct proportion to the concentration of the colored 
substance. Thus, the Intensity of the observed color Is directly proportional to the 
concentration of pigment In solution and directly proportional to the depth of the ob- 
served layer; and thus is actually proportional to the number of colored particles In 
the light path. 

For example; It would require a 20 mm. depth of- a 1% solution of a due to exhibit 
the same Intensity of color as 10 mm. of a 2% solution of the dye. Thus: 

(1) R x (20 mm.) x Cj (1%) = R 2 (10 mm.) x C 2 (2%) 

or 20 x 1 = 10 x 2 (see Fig. 16, p. 48a.) 

Obviously, If the light transmitted by a solution of unknown strength is compared 
with the light transmitted by a solution of a known strength, the concentration of the un- 
known can be calculated by means of this formula. Beer's Law, (for colorimeters) 
stated mathematically, is: 

(2) RjC^ R 2 C 2 

or C^/Cg = ^2'^1 

Here R, and R_ are the readings (depthB) of solutions of unknown and standard in 
the colorimeter cups when their transmissions match by visual observation. C^ and 
C 2 are their respective concentrations. This may be rewritten to solve for Cj, thus: 

(3) C x * (R 2 /R x ) x C 2 

C-, in this equation represents the concentration in the final colored unknown solu- 
tion. If only a small amount of material Is used in thiB test and it is desired to express 
the concentration In terms of 100 ml. , a volume correction factor (k) is Introduced Into 
the equation: 

(4) C t x (k) - C u = (R 8 /R u ) x C B x (100/V) 

In this equation C u is expressed (for example) in milligrams per 100 ml. of serum 
(mg.%); C is expressed in mg. of standard substance in the final volume of colored 
standard solution; and V represents the actual volume of original sample. For 



49 









example, if 1 ml. of a 1:10 protein-free filtrate is used In the analysis, V would be 
equal to 0. 1 ml. The above formula (4) assumes that the final volumes of the colorlme- 
tric solutions of the unknown and the standard are the same. In some determinations 
this may be not the case. A dilution factor (V /V ) Is accordingly introduced to correct 
for this difference. The final equation upon which all colorlmetric calculations in blood 
and urine analysis are based, thus becomes: 

(5) C u = (R 8 /R u ) x C B x (100 /V) x (V U /V B > 

in which V u and V , are respectively, the final volume of the unknown and the standard 
colored solutions. 100 ml. here Is the reference volume, and this Is the value often 
used for blood analysis. For urine it may be replaced by the 24-hour urine volume or 
one liter (1000 ml. ) depending on the particular application. 

Colorlmetric calculations are greatly simplified by choosing values of C B , V, and 
the colorimeter settings with that end In view. When the standard solution is set at a 
certain depth (R g ) the final calculation usually involves dividing the reading of the un- 
known into some simple factor which however, cannot be done mentally. Instead of 
using elaborate tables or calculating devices for this purpose, the expedient of setting 
the unknown at a given depth and reading the standard, enables one to complete the cal- 
culation mentally by a simple multiplication. For example, In a certain uric acid meth- 
od, setting the standard at 20 mm. requires the division of the reading of the unknown 
Into 80; while setting the unknown at 20 mm. requires only the multiplication of the 
reading of the standard by . 2. 

The standards employed, which are solutions of known concentration, may be of 
several types, their selection depending upon the nature and stability of the color devel- 
oped in the test. Theoretically, the mo Bt. desirable standard is one prepared by treating 
a known amount of the substance being analyzed with the reagent at the time of prepar- 
ation of the unknown. In this way, all of the slight variations of temperature, period of 
heating, and standing, etc. , are equalized . It is occasionally practical to use an arti- 
ficial standard made of more stable materials, such as a solution of one or more dyes, 
or a solution of Inorganic colored salts, or tinted glass plates. When using artificial 
standards it is necessary to check their equivalence frequently against the true color 
developed by known concentrations of the substance In question. In the case of colored 
glass plates used as artificial standards, the advantage of permanence may be offset by 
an imperfect color match. The colored plates should be standardized by spectrophoto- 
metry measurement to determine the plate equivalence in one's own laboratory. 

The colorimeter makes possible the quantitative comparison of the unknown and the 
standard solutions. It Is a precise optical device by which the Intensities of color In two 
solutions can be compared. If the intensity of color Is quantitatively related to the con- 
centration of a substance, colorlmetry may be employed for its quantitative estimation. 
The plunger type of Instrument Is best suited to general use in biochemistry. This in- 
strument has two plungers which dip Into the solutions contained in cups, and whose 



50 



( 



< 






relative positions may be regulated by racks and pinions. Light of uniform intensity is 
passed in parallel rays through the two fluids to be compared. The depth of one or both 
columns of fluid through which the light passes can be regulated at will by a vertical 
motion of the cups. The depths are so adjusted that the colors from the two solutions 
which are seen In a bisected visible field, appear equal . 

The colorimeter cups are of various types and sizes. The bottom of each cup is 
formed by a plate of clear optical quality glass. The tops may be flared out to serve as 
reservoirs for the solution displaced by the plungers when the cups are raised. These 
cups are optically perfect and such substitutes as small beakers cannot be used without 
greatly decreasing the accuracy which can be attained. Millimeter scales are arranged 
in connection with the cup supports so that they can measure the depth of fluid between 
the lower surface of the plungers and the bottoms of the cups. ' With the aid of verniers, 
measurement is precise to within 0, 1 mm. 

The visual field o f the instrument is a circle divided into two halves. The dividing 
line is formed by the ends of the prisms which transmit the light that has passed through 
the solutions. The optical path is arranged so that the light passing through the right 
hand cup appears on the left field and vice versa. When the two halves of the field show 
Identical color intensity, the conditions stated above in equations (1) and (2) are satisfied. 

Testing the Colorimeter : 

Before a colorimeter Is used each day and at frequent intervals during the day, it 
should be tested with regard to the zero points, the equality of illumination, and the 
equality of the quality of the transmitted color. 

Zero Point Test: The empty cups are first placed in position on the apparatus and 
carefully raised until the plungers come into contact with the bottom of the cups. Then 
the readings should be zero. If this is not the case, readings muBt be corrected for the 
zero point error. 

Equality of Illumination: Care must be taken that the illumination of the two fields 
is equal. Equal illumination can be secured best by using an even, ample source of light, 
preferably diffuse daylight or white artificial light. There are numerous satisfactory 
lamps on the market. Some colorimeters are provided with a light in the base, thus ob- 
viating the necessity of an outside source. 

Portions of the same colored fluid, usually the standard solution, are placed in each 
cup, rinsing the cups and the plungers, and taking care to avoid bubbles under the plung- 
ers. The cups are then set to have identical fluid depths and the light source adjusted 
until the two halves of the visual field appear Identical. After this has been done, color- 
imetric comparison may be carried out as outlined below. The colorimeter and Its light 
source should not be moved or altered after this light adjustment has been made. If a 
change does occur the light balancing should be rechecked before colorimetric compari- 
sons are made. 

51 






Colorlmetrlc Comparison : 

There should always be enough fluid to cover the lower ends of the plungers within 
the practical range of motion of the cups during a determination but the cups should never 
be so full that the liquid is driven over the top when the cups are raised to the reading 
point. 

After the above tests are completed, the standard solution is allowed to remain in 
one of the cups and the unknown is placed in the other, rinsing the cup and the plunger 
with portions of the unknown. The depth of the standard Is set at some convenient point, 
usually about 10, 15, or 20 mm, , and the height of the cup of unknown is adjusted until 
the two fields match. Several such adjustments are made, approaching the match point 
from both sides, and the average of such readings obtained is used for calculation. S 
is sometimes more convenient to set the depth of the unknown solution at a pre-deter- 
mlned point and then to adjust the depth of the standard solution to obtain the match. 

The stationary scale Is graduated in cm, and divided Into mm. All readings are 
made in mm. , e.g. , 2 cm. Is read 20 mm. The point at which the zero line of the mov- 
able scale would intersect the stationary one is taken as the reading. Decimal fractions 
are read at the point where two lines of the vernier and movable scales approximate 
most closely. 

Calculations are made according to Beer's Law which has been discussed above. 

Limits of Accuracy - Sources of Error \ 

Few observers can, with the usual colorlmetrlc instruments, match colors with an 
error of less- than 1%; for most analysts the error Is probably nearer to 2 or 3%. The 
accuracy is greatly influenced by characteristics of the Individual. Some persons are 
incapable of matching colors: others have difficulty only with certain colors. Adjust- 
ments should be made rapidly In order to avoid tiring the eyes. 

The range within which depth of solution is proportional to intensity of color Is 
quite limited. Standard and unknown can seldom be permitted to differ from one an- 
other by more than a factor of 2. If the difference is greater than this another standard 
must be used or the test must be repeated with a lower concentration (or higher, if 
necessary) of the unknown. 



52 



PHOTOMETRIC TECHNIQUES 



This procedure and technique differs from the method just described In two respects; 
(1) The depth of fluid examined Is not varied and then measured, but on the contrary, the 
depth is kept constant and the same for both standard and the unknown solutions. (2) The 
measurement which Is made Is not of depths giving equal light absorption, but an actual 
determination of the amount of light transmitted by the standard and by the unknown solu- 
tions. This measurement is made possible by use of a photo-electric cell, which gen- 
erates a current proportional to the amount of light striking the photo-cell. The current 
is then Indicated on the scale of a sensitive galvanometer. 

The relationship between the concentration of color in a solution and the amount of 
light transmitted through the solution can be treated in a mathematically rigorous fash- 
ion and this has been done in some of the reference books to which your attention has 
been directed. See p. 340. The biochemist and clinical technologist are usually interested 
In the simple comparison (in a quantitative way) of the amount of color developed by one 
or more unknown solutions with the amount developed by a solution containing a known 
amount of a standard substance. 

A diagrammatic outline of a simple filter photometer of a type commonly used in a 
laboratory is shown in Fig. 17, p. 53a. The white light from the bulb A passes through the 
color filter D. The light now passes through the test tube cuvet B containing the colored 
solution C. The emergent light strikes the photo-cell E, causing the generation of a 
current which is measured by the galvanometer G. The colored light filter may occupy 
the position shown or may be inserted between the cuvet B and photo-cell E. 

Briefly, the procedure in photometric analysis consists of the following steps: 

(1) Pure solvent (usually water) or a "reagent blank, " is used In a test tube cuvet. 
The cuvet is placed In position in the photometer and the rheostats R are adjusted so as 
to produce a reading of 100 on the galvanometer. The "blank" tube is then removed. 

(2) Without readjusting the rheostats R, the test tube containing the standard solu- 
tion Is placed In the photometer and the galvanometer deflection Is read and recorded. 
The "standard" tube is then removed. 

(3) Again without readjusting the rheostats, the test tube containing the unknown 
solution is placed in the photometer and the galvanometer is read and the result recorded. 

We have now a set of data consisting of the following: 

(1) T b = 100; (2) T s = X; (3) T y = Y 

where Tu is the per cent transmittance of the blank (arbitrarily 100); T is the per cent 
transmittance of the standard solution read from the galvanometer; and T u is the trans- 
mittance of the unknown solution, read from the galvanometer. 

53 













Simple rilter phoiomeler 




1 



I 



i 

ii 



Spectrophotometer (up to the photocell) 
Figure 17. Two types of photometers 



53* 






Filter Selection : 

The selection of the proper filter <D In the diagram) to use in a given analysis de- 
pends on the color which is being measured. Ideally, the filter should allow those wave- 
lengths of light to pass which the colored solution most strongly absorbs. Thus a blue" 
solution is blue because It absorbs most of the light In the red end of the visible spec- 
trum, and transmits most of the light In the blue end of the spectrum as diagrammed 
in Fig. 19, p. 54a. (A, B, C, D.) 

For the most sensitive analysis of this blue solution we would choose a filter which 
transmits light at the red end of the visible spectrum, I.e. , a red filter (indicated by the 
area with diagonal lines). It is never possible In practice to obtain filters which trans- 
mit exactly as indicated in the diagram. Instead the true graph of the transmittance 
(and absorbance) of a red filter would appear as shown by the curved solid line (X, Y,Z). 

Some filters have narrower transmittance bands and some have wider bands than 
this. In order for the solution to act in accordance with Beer's Law It Is necessary to 
have the filter transmit only those wavelengths of light which are absorbed maximally by 
the solution being tested. That is, those wavelengths on the flat part of the curve (C, D). 
This condition results in (a) the greatest sensitivity and (b) the narrowest range for 
any given method. 

In some instances it Is desirable to extend the range at the expense of Bensltlvlty 
and to some extent at the expense of linearity. This can be done by choosing a filter 
transmitting wavelengths between B and C In the example above. Further details re- 
garding the choice of a filter can be found in the reference books listed. 

Reagent Blanks: 

Since we are not working with ideal conditions and since reagents are not always 
pure, we often have a small amount of color or turbidity resulting In light absorption 
even when the reaction is carried out on distilled water or some other pure solvent. 
Such a preparation is called the "reagent blank. " There are two ways in which the 
blank can be utilized to correct for the impurities present and these will be discussed 
under GENERAL PHOTOMETRIC TECHNIQUE below. 

Beer's Law for Photometry: 

Let us assume that we have prepared a series of standards varying In concentra- 
tion along with a "reagent blank" and have recorded the corresponding galvanometer 
readings (which represent the transmittance in per cent), which are given here; 

Relative concentration Transmittance % 2 - log T = (D) 

100.0 0.0000 

1 80.2 0.096 

2 64.3 0.192 

3 51.5 0.288 

4 41.3 0.384 

5 33.1 0.480 
8 17.1 0.767 

10 1L0 0.959 

54 



100 



%T 




&l 1 1 1 



CONCENTRATION 



t.ol <- 



( 



CONCENTRATION 




CONCENTRATION 



12 






>■ 


.0 


#4 jf 


0.6 




0.6 
D 


/ 


0.4 


jr 


0.2 jf 


0.0 


/ > i i i i i t i i I i 


CONCENTRATION 



Figure 18. Types of photometric calibration -graphs. 









ioo 



TRANSMITTANCE 
% 




400 300 600 

WAVELENGTH OF LIGHT (mg) 



IOO 



70O 



Figure 19. Absorptance and Transmlttance spectra. 



54a 






There are a number of ways in which the relationship between concentration of sol- 
ute and galvanometer rcadtngs can be expressed. See Fig. 18, p. 54a. We note that in graph 
#1 (per cent transmittance vs. concentration) the result is a curved line. This type of 
relationship Is difficult to use routinely because it makes interpolation and extrapolation 
difficult and Inaccurate, Graph #2 (log per cent transmittance vs. concentration) results 
in a straight line with a negative slope. Graph #3 (per cent transmittance vs. concentra- 
tion) results in a straight line with negative slope if a special graph paper (semi -log) 
(ordinate plotted logarithmically and the abscissa plotted linearly) is used. Graph #4 la 
the easiest to use. It is a plot of the optical density (D) or (2 - log %T) vs. concentration. 
This plot results in a straight line with a positive slope . That Is, an Increase In con- 
centration results in an Increase in optical density . 

All of these graphs are expressions of Beer's Law, which can be expressed mathe- 
matically (for photometric analysis) as: 

(1) logde/y = -kc 

where 1^, is the intensity of transmitted light; 
Lj Is the intensity of Incident light; 
c is the concentration of the colored substance. 

This equation, stated in words, is that the logarithm of the ratio of the Intensity of 
transmitted light to the intensity of the incident light is inversely proportional to the con- 
centration of colored substance, k Is a constant whose actual value depends on the nature 
of the colored solution, the filter used, the depth of the solution, etc. 

In the experiment above, the incident light (the light transmitted in the absence of 
color in the solution — the "blank!') was arbitrarily set at 100% and therefore: 

(2) log (Ic/Io) = log (I c /100) 

Ig can now be termed the per cent transmittance and may be deslgna'ed with the symbols 
T or G (for transmittance or galvanometer reading),. 

Rearranging equation (2) 





log I c - log 100 = -kc 


or 


log 100 - log I c s kc 


or 


2 -log I c = kc 



If (2 - log I„) be given the name "optical density" and the symbol "D" then: 



c 

D = kc 



55 



It Is obvious that if experimentally a plot of D (optical density) vs. C, (concentra- 
tion) results in a straight line - usually passing through the origin (O.O), the results 
are In accordance with Beer's Law. Not all reactions giving colors will act this way , 
although a judicious selection of filters and/or the use of the more selective instru- 
ments Buch as the prism or grating spectrophotometer usually do result In a straight 
line graph (if the reaction conditions are correct). 

In ca s es of failure to obey the law , it will be necessary to use the plot of D vs. C 
(which will be a curved line in these cases), or to prepare a table derived from such a 
plot, to estimate concentration from the optical density. 

General Photometric Technique : 

1. Selection of filter: Selection will depend on the color developed In the determina- 
tion. The wrong filter will give incorrect results. 

2. Warm up : Allow the instrument to come to a "steady-state." This will take 15 
to 20 minutes. 

3. Adjust galvanometer to read zero : Adjust the zero point reading when no light 
Is striking the photoelectric cell. This is very Important because the 100% setting is 
not really 100% and no other reading will be correct unless the zero setting has been 
accurately made. 

4. Set the "blank" to read 100 : Insert the cuvet or selected test tube containing 
the "blank" solution (read further below) and adjust the light intensity so that the galva- 
nometer reads 100%. 

5. Read the standard : Remove the blank tube, insert an identical cuvet or test 
tube containing the standard and read its trans mittance T g , in per cent. 

6. Read the unknown: Remove the standard tube, insert an identical tube or cuvet 
containing the unknown solution, and obtain its transmittance T in per cent. 

7. If the photometer was adjusted to read 100 using a pure solvent as the "blank, " 
the "reagent blank" must now be read and its transmittance recorded as T^ in per cent, 

"Blank" definitions: 



( 



It will be noted that the term "blank" has been used in several different ways in the 
above discussion. For the purpose of clarification the following definitions should be 
carefully noted. 

"Optical Blank" is the solution used to set the photometer at 100 per cent trans- 
mittance . 

"Reagent Blank" is the solution obtained when the colorimetric reaction Is carried 
out on pure water rather than on a solution containing the substance analyzed for. 

"Zero-time Blank" is the solution obtained when no time at all Is allowed for a re- 
action whose optical density increases with time. This Is commonly used only In cases 
of estimations of enzyme activity, when there is present in the serum some constituent 
which is being formed from a substrate by enzymatic activity. 



56 









4 should be noted that the reagent blank and the zero-time blank can be utilized as 
optical blanks. It is of some advantage to do so, because by such a procedure, the blank 
value Is automatically subtracted from the unknown and avoids an extra calculation. 
See below for the details of such calculations. 

Photometric Calculations: 

As in visual colorimetry we are determining the ratio of the concentration of color 
in the unknown as compared to that In a standard and therefore the general formula Is 
very similar, remembering that the t ransmlttance must be converted to optical density 
before inserting in the formula. 

Visual colorimetry formul a Photometry Formula 

C u = <R 8 /R U >C S C u = <D u /D fl )C a 

Also note that the readings (R) In colorimetry are inversely related to the concentration 
while in photometry the densities CD) are directly related to concentration. 

The oomplete formula for photometric calculation la analogous to the colorimetric 
formula p. 49. 

(3) C u = (D u /D fl ) x C 8 x (100/V) x (V U /V B > 

Since in most photometric methods, V u /V a is 1, the last term usually cancels out. 

Whenever blanks with an optical density greater than 0, (T = ^100) are obtained, 
(such as reagent blanks or zero-time blanks read against a pure solvent optical blank) 
the optical density of the blank la subtracted from the unknown and standard densities 
before inserting the values into the formula. 

When it has been shown that a result Is readily duplicable for long periods of time, 
it Is possible but not recommended to dispense with the use of standards and to use the 
relationship, D s kc, where k Is the constant relating density and concentration by a 
combination of factors such as the reaction characteristics and the characteristics of 
the color and the filter used, etc. Thus, In use, k is determined by the use of standard 
solutions and thereafter the density values of the unknowns is multiplied by k to obtain 
the final result, say in mg. per 100 ml. of blood. 

This procedure la somewhat dangerous and should not be resorted to unless abso- 
lutely nceessary. ft is used in cases in which scarcity or instability of the standard 
material limits the supply as for example a pure bilirubin, urobilinogen, etc. 

Some photometers, such as the Klett, have a scale reading directly in optical den- 
sity units. Unless this is the case, the log of the ratio must be calculated for Ig/I or 
a table of D vs. T values may be used. See Appendix for such a table. 

57 



In the foregoing photometry discussion, It was assumed that a constant depth of sol- 
ution was being used. This laboratory uses especially selected test tubes as absorption 
cells In most cases. Although these do not have a uniform depth of solution for all parts 
of the tube, still the effective depth Is the same for the matched tubes. Since test tubes 
inevitably are somewhat oval rather than round, it la imperative that they be positioned 
In exactly the same way each time they are used as well as when they are calibrated. 
See p. 330 for a note on the Calibration of Test Tube Cuvets. Calibration can be avoided 
If the same test tube (In the same position) Is used for the reading of the standard and 
for the unknowns as well as for the blank setting or reading. However, in some analy- 
ses, this procedure Is Impractical. 

Photometric instrumentation: 



In the discussion above, a simple single photo-cell photometer Is described. There 
are, however, a large variety of types of Instrument available on the market. They 
may be classified as follows: 

The essential parts of any photometer are: 
{1} A source of light equipped with 

(a) a means of adjusting the intensity of the light falling on the solution and 

(b) a means of stabilizing the Intensity of the light so that it remains constant 
for long periods. Since the emissivity of a tungsten lamp is proportional to the fourth 
power of the absolute temperature, the power supplied to the lamp must be very closely 
regulated. 

(2) A means of wave-length isolation which may be by : 

(a) Color filters - plates of colored glass with definite absorption character- 
istics, 

(b) Interference filters - plates of glass with half-wavelength reflection. 

(c) Prism or gratings - used in spectrophotometers. These can be rotated 
to select a given narrow band of wavelengths. 

The usual way of expressing the band width of a filter or other device for Isolating a 
band of wavelengths Is to state the band width between the points giving 50% of the 
transmlttance of the peak. Using this notation, Corning glass filters have a band width 
of 50 to 150 mu; some interference filters have a width of 10 to 40 mu; for the Coleman 
spectrophotometer the width Is about 35 mu; for the Bausch and Lomb Spectronlc 20 - 
about 20 mu; and for some more precise instruments as small as 1,0 or 0.5 mu 
depending on the wavelength. These Instruments may be equipped with quartz optics to 
enable their use In the ultra -violet region of the spectrum. 

(3) Cuvets - these may be 

(a) very precise square cuvets 

(b) especially selected test tubes - less accurate, more convenient 

(4) Photo-cell (s) may be of two types 

(a) Barrier layer cells - these generate their own current but require a 
sensitive galvanometer since the current cannot be readily amplified. 

(b) Photo-emissive cells - which change resistance upon exposure to light 
and require an external current source. The current in this case may be readily ampli- 
fied, thus a less sensitive galvanometer is required. 



C 






The available Instruments may also be classified as single cell or double cell in- 
struments. The Blngle cell type 1b the simple type described above, p. 53, The double 
cell type consists of two closely matched photocells which can be balanced against each 
other so that no current passes when the reading Is made. The Klett-Summerson in- 
strument is of this type. The reading is made in optical .density units from a dial con- 
nected to the balancing rheostat circuit. 

Flame Photometry: 

This la a very specialized application of photometry in which a finely divided aero- 
sol spray of eample is passed into a flame. The heat excites the elements present so 
that they emit light. This light is filtered by use of glass color filters or by prism or 
grating arrangements and the wavelengths of Interest are allowed to fall on a photocell, 
The light produced is proportional within narrow limits to the amount of a particular 
element present. 

This method is commonly used for sodium and potassium in many laboratories. To 
compensate for some of the many variables, the double cell method is used in some of 
the instruments. 

In the double-cell (or internal-standard) method, one cell and filter combination Is 
responsive to say sodium emission, and another cell -filter combination is responsive 
to lithium emission. Since lithium does not exist in biological fluids It can be added to 
the same concentration in known and unknown solutions. When this is done the readings 
of the Instrument are responsive only to the ratio of sodium to lithium and since any 
factor decreasing sodium emission will also decrease lithium emission, the reading Is 
quite stable. See further under Na and K. 

In the use of all these Instruments it should be remembered that ordinary white 
light is actually made up of all the colors in the rainbow. By filters or by gratings or 
prisms white sunlight may be separated into its component colors. One thing these 
colors all have In common Is thatthey travel, in waves. The important difference be- 
tween the colors Is that each one has a different wavelength (the distance between the 
crest of one wave and the crest of the next). When all the colors in white light are 
Bpread out and arranged by wavelength (as may be done in a rainbow or by a priBm) , 
they make up a continuous band. At one end of the band is violet, with a wavelength of 
400 millimicrons (mu) which Is about 16 millionths of an inch. Jtextin order come 
indigo, blue, green, yellow, orange, and finally red light, whose wavelength is com- 
paratively long (700 mu). 

These wavelengths from 400 to 700 mu (the so-called "visible" range) are the only 
ones the human eye can see. Light with wavelengths outside of this region does not 
promote a visual response of color and brightness In the human eye. At wavelengths 
Just below our area of visibility lies the ultraviolet region. Above the red area of 
light lies the infrared region. 



69 



Chemical analysis may be done by use of a spectrophotometer because the atoms 
Id a molecule resonate to certain wavelengths of ultraviolet, visible, or Infrared radia- 
tion. The wavelengths which cause this vibration are absorbed; the rest of the wave- 
lengths are transmitted through the sample. Now If we make a graph showing hovyum'ch 
of each wavelength a particular chemical compound absorbs, we have its "absorption 
spectrum." Fig. 17, p. 53a shows how the spectrophotometer measures the amount 
of a particular color (wavelength) of light absorbed or transmitted by the sample sub- 
stance. By making a series of such measurements and using different wavelengths of 
light, the analyst can draw a curve which shows the exact location and degree of the 
absorptions of the sample over a wide range of wavelengths. This curve is the "absorp- 
tion spectrum. " 



( 



IF a series of measurements are made with different concentration at a certain 
specified wavelength the results obtained are called a "calibration curve". This can be 
used in quantitative analysis as discussed above on page 65. 

























































60 



GASOMETHIC ANALYSIS 



In the study of gases and gas volumes we must constantly be aware of the effects 
of temperature and pressure and their changes from one determination to the next. 
The volume of any gas depends upon the temperature and pressure to which It is sub- 
jected and the volume of a gas may vary widely with changes of temperature or pres- 
sure. It Is necessary, therefore, in order to compare a volume of a gas measured 
under one temperature and/or pressure with a volume of gas measured under a second 
temperature and/or pressure, to have a set of uniform correction factors. 

The Standard Gas Conditions are as follows: 

1. Atmospheric pressure at sea level (780 mm. column of Hg). 

2. The temperature of water at Its freezing point (0° centigrade or 273° absolute). 

Gasometric methods are those which depend upon the measurement of a gas as the 
final step In the analysis. The measurement must take Into consideration the so-called 
gas laws . 

The gas laws governing the behavior of the gases are known by the names of the 
men who first clearly formulated them. 

Boyle's Law (1660) 

Boyle's law states that If the temperature Is constant the volume of a gas varies 
Inversely proportionate to the pressure. 

Boyle's law may be expressed as follows: 

|i-.^2- OTPy^ m P 2 V 2 or V i: P 2 .= Vjj*! 

The table below will aid In demonstrating the Inverse relationship between volume 
and pressure as defined by Boyle's. 

Pressure Volume 

P, 1 atm. Vj 18 cc. ft is easy to. see that as the 

P« 2 atm. V 2 8 cc. pressure increases the volume 

P 3 4 atm, V„ 4 cc. decreases; and as the pressure 

P. 8 atm. V. 2 cc. decreases the volume Increases 

P, 16 atm. V R lcc. (T being K). 

5 



61 






Example of use of Boyle's Law 

A volume of a gas measured at 740 mm. pressure Is 200 ml. 

(a) Calculate the volume of gas at standard pressure. 

(b) Calculate the volume of gas at 610 mm, pressure. 

W Pi = 740 V a - 200 x fg . 194.7ml, 

P 2 = 760 
Vi = 200 ml, 
V a - unknown 740 x 200 m 760 x V g 



Student exercise: 
Solve part (b). 

Charles' Law (1801) 

Charles' law states that If the pressure remains constant the volume of a gas will 
vary directly as the absolute tempera tu re . 

Charles' law may be expressed as follows: 

V 2 - T 2 

~ ■ T or V 2 .Ti s Vi ,T a or Vi: T x = V 2 : T 2 

v l *1 

Therefore, It can be seen that as the temperature Increases the volume In- 
creases, and as the temperature decreases the volume decreases (P being K). 

Example of use of Charles' Law 






V 2 ■ 148000 s 194.7 ml. 




760 





















A volume of gas measured at 70°C. Is 300 ml. Find Its volume at: 

(a) 0°C. 

(b) 100°C. 

A T l = 70°C. 
T- s 0°C. 
Vj = 300 
V« = Unknown 



Student Exercise: Solve part (b) 






I2. = l2_ OT V, 
V l T l * 


Ti = V x • T 2 


Vo 273 

—A v« 

300 343 2 ' 


343 z 300 . 273 


300 x 273 
V 2 a 343 


300 x 273 
V 2 * 343 


Y . 81900 
2 " ti« 


V 2 ■ 238. 8 ml. 



( 



62 









s& 



& 



s& 



*>< 



^r\ «^M 



aOV 



& 



-lOO" 



- 50° 



O* 
•10* 



^40° 



-47y 



w 



-212° 



-122° 



32° 
+14 



—40° 



i459" 



^ / 



*r\ 



-373° 



-323* 



-273' 
■2631 



-223" 



- 0' 



s/ 



FREEZING POINT 



—ABSOLUTE ZERO 



<J 



Figure 20. Comparison of thermometer scales. 



62ft 



( 



It has been demonstrated that all gases expand to the same extent when exposed to 
the same Increase In temperature (P la K). It Is further shown that a 1°C. rise In 
the temperature of a gas will expand the gas by 1/273 part of Its volume at 6°Cj a rise 
of 2°C. will Increase the volume by 2/273 of Its volume at 0°C; or a 273 ml. volume 
of ft gas would become 274 ml. and 275 ml. respectively. Also a i°C. fall In temper- 
ature of 273 ml. of gas will become 272 ml. or be reduced by 1/273 part of Its volume 
at 0°C . For the purpose of gas calculations we use a "Absolute 1 ' or "Kelvin" scale of 
temperature at which 273°A = 0°C. 

The value of 1°C. and 1°A is the same (Fig. 20, p. 62a) except that the freezing point 
on the absolute scale la 273°A and boiling point is 373° A as opposed to 0°C. to 
100°C. on centigrade scale. 

It is the absolute temperature that is always used when calculating to correct for 
temperature changes In tae gasometrlc analysis. 

It will be of value to the technician to know the general formulas for conversion 
from the Fahrenheit to the Centigrade scale and from the Absolute to the Centigrade 
as seen in Figure 20. 

212°F - 100°C = 373°A 

Conversion of Fahrenheit to Centigrade 

C s (F-32) x 5/9 or C: X^22_ 

1.8 

C = (212-32) x 5/9 C a 212 ~ 32 

1.8 

C - 180 x 5/9 = 100 C = -IS® = 100°C. 

1.8 

Conversion of Centigrade to Fahrenheit 
F - (100 x 9/5) / 32 F = (100 • 1.8) / 32 

F = (20 X 9) / 32 F = 180 / 32 

F = 180 / 32 s 212° F = 212° 

Centigrade to Absolute (or Kelvin) 
°C / 273 = °A 



63 



If Boyle's and Charles' Laws are combined: 

V oC(l/P) x T 

and In general: V : nRx T/P or PV a nRT 

where n represents the number of moles of gas prosent and R la the molar gas constant, 
the numerical value for which depends upon the units In which the pressure and volume 
and temperature are expressed. For pressure In atmospheres , volume In liters and 
temperature in degrees absolute , R Is equal to 0. 08205 Uter-atmospheres per mole 
per degree. 

Thus if we know the volume In liters , the pressure tn atmospheres , and the temp- 
erature in absolute degrees, we can calculate the number of moles of gas present . 

Dalton's Law 

p t = p x /p a /p 3 ^p 4 / 4 P n 

Dalton's Law states that the total pressure of a gas mixture Is the sum of the pres- 
sures of the Individual gases la the mixture, 

Henry's Law 

Cj = kP 

Henry's Law states that the solubility of a gas In a liquid is proportional to the 
pressure of the gas. Each gas has Its own solubility coefficient k. Temperature In- 
c reases will decrease the solubility of gases in liquids so k will vary with the temper- 
ature. 

If the amount of a gas present is to be expressed In terms of a volume , the tem - 
perature and the pressure must be stated . The usual values chosen for a standard 
temperature and pressure are: 

0°C, (273° absolute) 

760 mm. Hg (mercury) (one atmosphere) 

When it is necessary to convert a volume of gas at one temperature and pressure 
to the volume at another temperature and pressure the following formula is useful; the 
subscript "1" refers to one set of conditions and "2" to another, 

PlVl . p 2 v 2 



( 



T l T 2 



( 



64 



Example ; A gas measured at 20°C. and at 740 mm. Hg. pressure had a volume 
of 30, 7 ml. What would be its volume measured under standard conditions? 

740 x 30.7 - 760 x V ? 
20 / 273 273 

V 2 s (740/760) x (273/293) x 30.7 

s 0.974 x 0.932 x 30,7 

r 27. 3 ml. 

Kote that the temperature correction factor and the pressure correction factor can be 
handled separately. 

Water Vapor Pressure Correction: 

When the volume of the gas is measured over water, part of the pressure (and part 
of the volume) Is due to water vapor. The easiest way to handle this correction Is to 
subtract the partial pressure of water vapor at the observed temperature from the total 
pressure to give the pressure due to the dry gas. 



Temperature Vapor Pressure 


Temperature Vapor Pressure 


°C. 


mm. Ha. 


°C, mm. Hk. 


16 


12.8 


26 25.2 


16 


13.6 


27 26.7 


17 


14,6 


23 28.3 


13 


15.6 


29 30.0 


19 


16.6 


30 31.8 


20 


17.6 


31 33.7 


21 


18.7 


32 35.7 


22 


19.8 


33 37.7 


23 


21.1 


34 39.9 


24 


22.4 


35 42.2 


25 


23.8 




Example: A gas sample Is measured at 27°C. , at 750 mm. Hg. pressure, and 



over water and Its volume Is found to be 34. 2 ml. Calculate the volume of the dry gas 
at standard conditions. 

At 27°C. the vapor pressure of water Is 26. 7 mm. Hg. (see table). Then 

using the formula: 

PlVl _ P 2 V 3 
Tl - T 2 



66 



(750 - 26.7) x 34.2 760 X Vg 
(27 / 273) " 273 

V 2 - (723.3/760) x (273/300) x 34.2 

- 0.962 x 0.010 x 34.2 

s 29.6 ml. 

Molar Gas Volume ; 

Squat volumes of all gases contain the same number of molecules. Thus each gas 
has a molar volume of 22. 4 liters (at standard temperature and pressure). Thus we 
can calculate moles (and grams) from volumes and vice versa. 

Example : Convert 29.6 ml, CO« (M. W. 44) at standard temperature and pressure 
to moles and also to grams. 

29 - g "»1- = 0.00132 moles or 1.32 mil 
22,400 ml. 

1.32 x 44 a 68.2 mg. or 0.0582 g . 

There are TWO GENERAL METHODS OF GAS ANALYSIS 

(1) Volumetric - "volume measurement" 

The pressure Is "set" (usually at atmospheric pressure). 
The volume is read (and also the temperature). 

(2) Manomctrlc - "pressure measurement" 

The volume Is set (usually at 2.0 or 0.8 ml.) 

The pressure is read (usually In terms of mm. Hg.) (and the temperature 

is also read). 

For further details of specific uses of gas analysis, see Carbon Dioxide, p. 129. 






URINE COLLECTION AND PRESERVATION 

In this work 24-hour samples of urine are usually used. Quantitative determina- 
tions of the constituents of random voidinga are of no value or significance In the inter- 
pretation of metabolic processes. It is only when all urine produced by the kidneys 
over a known period of time is analyzed that conclusions can be drawn as to what the 
body is doing with various foodstuffB. The shortest period of time that is usually practi- 
cable for this purpose is an hour, but a twenty -four hour sample 1b usually preferable. 

Collection of 24-hour urine specimen: 

It is necessary to have a bottle of about two liters capacity, with a stopper or cover. 
The bottle should be clean. In it is placed 1 to 2 ml. of toluene to act as a preservative. 
(In some cases the use of toluene must be avoided) . 

At a convenient time in the morning (say 7 o'clock) the bladder is emptied and the 
specimen is discarded. This voiding represents urine produced by the kidneys before 
the time of collection. Thereafter all urine voided up to and including the 7 A.M. void- 
ing (or the time corresponding to that of the discarded specimen of the previous morn- 
ing) is placed in the bottle, which should be kept in a cool place. Analyses should be 
carried out as soon as possible after collection of the specimen. 

If It Is not possible to collect the entire 24-hour specimen in a single container, 
all portions of it should be poured Into a single container and mixed well to make it 
homogeneous before any sample is removed. Thereafter the specimen should be stirred 
up well each time a sample is removed. Otherwise, more or less of the sediment will 
be taken out than should go with the removed portion, the composition of the removed 
portion and of the remaining portion being changed thereby. 

Measurement of volume: 



Since the amount of a constituent In the whole specimen Is found by calculation 
from the amount determined in an aliquot, it is necessary to know the exact volume 
of the 24-hour specimen. Hence the volume should be carefully measured before any 
other determination Is made. Sometimes it is necessary to determine the separate 
volumes of the urine excreted in the first 12 hours and that excreted during the second 
12 hours. Volume measurements can be carried out during collection of the specimen, 
but none of the specimen(s), should be discarded. Specimens for routine urine analysis 
with no quantitative determinations may be random, although it Is much preferable to 
obtain morning specimens for this purpose. 

Preservatives: 

1.. Toluene Is a very acceptable preservative for urine for general use. 

2. Formaldehyde or oxalic acid may be used In Bpeclal cases. 

3. Sodium carbonate 5 g. is used in addition to petroleum ether or toluene for the 
preservation of the biliary and porphyrin pigments. 

67 



4, A few ml. of glacial acetic acid may be used In cases In wlilch maintenance of 
an acid pH Is required such as Addis Counts and Calcium In urine. 

5. The beet method Is storage In a refrigerator but this Is not always practicable. 

Changes during storage of urine: 

1. Bacterial action . Since urine Is usually not collected aseptlcally, It la not 
sterile. However, If clean containers are used, little change will be noted In a 24- 
hour collection period especially if it is kept cool and stoppered during the collection 
period. 

Bacterial action on urea : 

This results in the formation of ammonia, carbon dioxide, and a change in 
pH. Ammonia (and its nitrogen) may be lost to the air. If the urine becomes alkaline, 
calcium and magnesium phosphate salts will precipitate. 
Bacterial action on glucose : 

In a specimen containing glucose, the above actions may be accelerated and 
the glucose may disappear due to Its utilization by the bacteria. 

2. Uric acid and urates may precipitate on allowing the urine to stand, especially 
in the refrigerator. Before analysis, all sediments must be well suspended and a 
sample taken of the well mixed specimen. Other changes may occur, some of which 
are mentioned under the particular method when necessary. 

FECES COLLECTION 



For qualitative tests, such as for occult blood, only a portion of the stool collec- 
tion need be sent to the laboratory. However, It is important that this portion be 
carefully selected, preferably by the physician ordering the test. 

For quantitative tests, and especially for balance studies, complete collection of 
all stools passed is necessary. The stool should be collected In such a way that urine 
does not become mixed with the sample. In most cases, it is possible by inspection to 
detect urinary admixture. Considerable amounts of chloride also indicate urine con- 
tamination since feces contain little chloride ion. 

Marking of feces : 

In balance studies it is sometimes desired to collect all feces formed during a 
given period of time, say 3-4 days. 

A neutral substance such as charcoal, carmine, gentian violet in amounts of 5 
grains (300 milligrams) may be given in a gelatin capsule with the first meal of a given 
experimental period and again with the first meal after the experimental period has 
ended. Thus the first feces associated with the test period will be that first marked 
with the dye. The first feces after all test period samples have been collected will be 
marked with dye and should be discarded. Save the first marked specimen and all 
stools thereafter up to, but not Including, the next marked specimen which should be 
discarded. 

68 









Sampling of Feces : 

Since the constitution of feces la variable the problem of sampling the total col-' 
lection ia critical. 

1. Dry mixing and sampling - the entire sample ia dried, ground, and then well 
mixed. A properly withdrawn aliquot la then analyzed. 

2. Wet mixing and sampling - this procedure uses a Waring blender to produce 
a homogeneous pipettable suspension. 

Procedure : 

1. The sample la collected into a large Jar with a wide mouth, up to three days. 
The sample ia always kept in a refrigerator. 

2. At the end of that time, approximately 1500 ml. of water la added, and the 
lid of the jar stoppered tightly. 

3. Add 1 ml. of capryllc alcohol to prevent foam, and shake vigorously. 

4. Pour portions gradually Into a Waring Blendor and homogenize completely. 
6. Add all the homogenized portions to a 2000 ml. graduated cylinder and dilute 

exactly to 2000 ml. 

6. Pour all Into a large bottle or flask, mix by shaking and pour aliquots Into 
two 250 ml. brown bottles. Stopper well. 

7. Deep freeze until used for analysis. 

8. For individual analyses, see the appropriate determination. 

9. For almost all kinds of analysts, aliquots may be measured accurately from a 
Yale BD Lok-Syrlnge, which delivers accurately to 0.1 ml. If properly handled. 

Calculations : 

If the collection period was three days, for any given constituent: 

g/day . (g/ml.) 299$. 

3 

Precautions; 

1. Fill bottles only 2/3 full, as in freezing the expansion will cause the bottles 
to crack If full. 

2. Steps 4, 5 and 6 are critical. Unless the specimen 1b homogenized well enough 
to be pipetted accurately, results will be unreliable. No discrete particles must re- 
main In the final mixture. 

Preservation of feces: 

Specimens should be analyzed as soon as collected to avoid the necessity of add- 
ing preservatives. 

If feces must be preserved it is best to treat them as indicated under wet mixing 
above . 



69 



COLLECTION OF BLOOD SPECIMENS 

The clinical analysis of blood (as well as of urine and other biological materials) 
really starts with the patient, m order to obtain as reproducible results as possible, 
most blood samples are collected from patients In a "post-prandial" state. This is the 
time at about 12-14 hours after food has been taken, usually In the morning before 
breakfast. ThiB procedure minimizes the effects due to digestion and absorption of 
foodstuffs on the one hand, and the effectB of "starvation" (i.e. , mobilization of tissue 
fat and carbohydrate) on the other. In some analyses it Is also important to limit the 
motor activity of the patient (e.g. , by keeping him In bed) before the sample is taken. 

There are a number of routes by which blood samples may be taken. They will be 
noted here, together with a brief outline of the essential points of the procedure. 

I, Capillary Collection : 

Capillary blood is most frequently obtained from a finger or thumb, ear lobe or 
heel (In Infants). The site is first cleaned with alcohol or acetone, the solvent Is al- 
lowed to dry and the site is then pricked quickly with a sterile needle. A freer flow of 
blood Is obtained if the area is warmed for 10-15 minutes before a collection, and If 
this is done, the characteristics of capillary blood closely approximate those of arter- 
ial blood, especially when taken from finger or thumb. The prick should be deep 
enough to insure sufficient flow for the collection. 

This procedure Is usually used when it is desired to collect 0.1 to 0.2 ml. of blood 
although up to 2 ml. can sometimes be obtained by this method. 

The blood is usually collected directly into plpets calibrated "to contain. " The 
bore is quite small so that the blood runs Into them without aspiration if they are kept 
horizontal. Thi6 Is true only if they are scrupulously clean. 

The blood Is allowed to flow into the pipet until it is filled to just above the calibra- 
tion mark, the upper end Is closed off with the top of the forefinger. After adjusting 
exactly to the mark by tipping the end of the pipet to a filter paper, or to the finger, the 
pipet Is wiped clean and the contained blood is allowed to run Into a tube containing 
water or other diluting solution. By alternately blowing and aspirating, the blood is 
washed from the pipet by the solution. Various types of solution may be used depending 
upon the analysis to be made. See deproteinization, p. 76. 

n. Venous Collection : 

Since usually more than one determination is to be carried out, venous collection 
Is preferable. The blood is usually withdrawn from a vein in the antecubltal space of 
the arm. The syringe used should be large enough to collect the entire specimen. In 
most cases, the tourniquet used to demonstrate the vein should be removed before the 
blood sample is collected since stasis of the blood alters some of the clinically signi- 
ficant blood values. For details and illustrations of the technic of venipuncture see 
The Handbook of the Medical Corps, U.S. Navy and also the Serology Manual. 

After the sample has been obtained and the needle removed from the vein, pressure 



70 



( 






should be maintained on the site to prevent peri-vascular hemorrhage. The blood in 
the syringe is then promptly transferred to plain tubes, or to tubes containing anti- 
coagulant (eee below), 

IK. Arterial collection: 



Collection directly from arteries is rarely made. It may be taken for blood-gas 
studies. It is usually withdrawn from the radial or brachial or the femoral artery and 
usually under local anesthesia. 

Prevention of Hemolysis : 

In many determinations it Is preferable to use serum. Certain determinations can- 
not be run if there Is even a small amount of hemolysis. It is worthwhile, therefore, to 
note the steps that are necessary to prevent hemolysis: 

1. The syringe and needle used must be dry. 

2. The needle used must be of a sufficiently large bore to allow the blood to run 
through it without being forced to too high a velocity. A 20-gauge needle Is satisfactory. 

3. Blood should be allowed to flow Into the syringe; one should not pull back hard 
on the plunger. 

4. The needle should be removed before the blood is transferred into the clean, 
dry test tube. The blood should be allowed to run gently down the wall of the tube and 
should not be squirted into it. 

5. The tube should be allowed to stand quietly for 10 to 15 minutes without being 
agitated. It should not be allowed to roll around on the bottom of a tray. 

The same steps can be followed In obtaining oxalated blood except that It Is neces- 
sary to stopper the tube and invert gently a number of times as soon as the blood Is 
placed in it, to dissolve and mix the oxalate. 

Centrifugatlon: 

When centrifugatlon Is to be carried out the following principles must be observed 
to avoid damage to the centrifuge or to the operator. 

1. The principle of "balance" must be observed. Tubes and cups of equal weight, 
shape , and size should be placed in opposing positions In the centrifuge head. 

2. Tubes should be buffered by rubber cushions in the brass cups, and additional 
cushioning may be obtained by using water in the brass cups. 

3. The glass tubes used should be inspected to insure that they have no cracks or 
chips. Heavy stoppers, (either rubber or glass) should not be used. Corks are prefer- 
able In most cases. 

4. When these precautions have been observed, the lid of the centrifuge is closed, 
and the machine is slowly speeded up until the desired speed is attained. After the re- 
quired length of time, the power is cut off and the centrifuge allowed to come to rest 
by itself. Rapid slowing by a brake can stir up precipitated material. 



71 






Choice of sample : 

When carrying out blood nnalyslo wo may choose to use whole blood, scrum, or 
plasma. Serum Is preferred to whole blood or plasma whenever Its use is practical. 
There are a few general principles to be considered in the choice of the sample to be 
analyzed. 

1. If the substance analyzed for is evenly distributed between red blood cella and 
plasma (or serum) it does not matter which is used. If a capillary collection is done, 
whole blood may be more convenient. 

2. Some substances present in erythrocytes may adversely affect the accuracy of 
the determination by Interfering In the reaction. In such cases it is better to use serum 
or plasma. 

3. In some cases the clinically significant variation in concentration occurs only 
in the serum or plasma, for example in serum chloride, which varies much more with 
disease states than does whole blood chloride since the red blood cell chloride concen- 
tration Is relatively stable. 

4. If there are great differences in concentration between rbc and plasma, varia- 
tions in hematocrit will greatly affect the results. 

5. It is easier to avoid hemolysis in the production of serum, but when the separ- 
ation and analysis must be made quickly plasma is usually chosen. 

Preparation of blood for analysis: 

After the blood has been drawn from the patient, it is treated in various ways to 
obtain suitable samples for analysis. 

1. Serum: 
Serum should be used wherever it is practicable. To obtain serum the needle 

is removed and the blood in the syringe is transferred without undue trauma to a clean 
dry test tube which can be centrifuged. The tube is allowed to stand quietly without 
agitation for 15-30 minutes. The clot Is then loosened from the wall of the test tube 
with a glass rod or applicator stick and the tube Is centrifuged for about 5 minutes at 
about 1500-2000 rpm, stoppered with a light rubber stopper or a cork. The serum is 
removed by the use of a rubber bulb fitted to a glass tubing, the end of which is drawn 
out to a coarse capillary. If any rbc are Inadvertently included in removing the super- 
natant, the serum should be recentrifuged before sampling. 

2. Plasma: 
If plasma is to be obtained an anticoagulant must be promptly mixed with the 

withdrawn blood to prevent clotting. Most of these act by combining chemically with 
the Ca^ of the blood to prevent Its participation in the clotting mechanism. Oxalate 
and fluoride act by forming insoluble calcium oxalate and calcium fluoride. Citrate 
and EDTA (ethylene-diamine-tetracetlc acid) act by forming un-lonized calcium salt 
complexes; heparin acts (a) In the first stage of coagulation to prevent the production 
of plasma thromboplastin, (b) in the second stage to inhibit .the formation of thrombin 
from prothrombin (and apparently as an antl-accelerin) and (c) In the third stage to 
Inhibit the action of thrombin on fibrinogen. Heparin acting alone is not an anticoagu- 
lant. It acts in conjunction with certain albumin fractions of plasma. Due to lack of 
this albumin factor rabbit blood will clot in the presence of large amounts of heparin. 



72 



Anticoagulants : 

1. The anticoagulant most commonly used In the clinical chemistry laboratory Is 
potassium oxalate , K 2 C 2 0.- H„0. This salt can be made up in a 30% solution by dis- 
solving 30 g. In enough water Fo make 100 ml. of solution. One-tenth milliliter of this 
solution contains 30 mg. , an amount sufficient to prevent the coagulation of 15 ml. of 
properly drawn blood. If smaller samples of blood are anticipated, correspondingly 
lesser amounts of the anticoagulant solution should be employed (2 mg. of K a C,0 4 * H«0 
per milliliter of blood). The proper amount of solution is introduced into the bottom of 
a clean, dry, test tube which Is then rolled on its side on a hot plate until water has 
evaporated, leaving the oxalate as a loose salt deposit scattered over the walls of the 
tube. A high temperature should be avoided in order to keep from converting the oxa- 
late to carbonate. Such tubes can be prepared in quantities, stoppered, and stored 
indefinitely. 

2. Mixed oxalate (for hematology). The above salt makes the plasma somewhat 
hypertonic and causes some water to be drawn out of the cells with consequent dilution 
of plasma constituents and shrinkage of the cells, if It is desired to prevent such shift 
of water and if the presence of ammonium ions does not interfere with subsequent uses, 
a solution of ammonium oxalate, (NH J 2 C 2 0^- H„0, and potassium oxalate can be made 
as follows: Dissolve 6 g. of (NH.) C 4 ' H 2 and 4 g. of KgC-O^ H g O in water, dilute 
up to a volume of 100 ml. , and mix. one-tenth ml. of thiB solution contains enough 
oxalate for 5 ml. of blood. 

3. Fluoride (in the form of sodium fluoride), NaF acts as an anticoagulant but Is 
required in much larger amounts (100 mg. /10 ml. of blood) than is necessary for oxa- 
lates. K is commonly used in combination with thymol and oxalate whenever blood must 
be preserved for later analysis. Fluoride inhibits the action of certain enzymes (such 
as urease) and this effect must be kept in mind in certain analyses. The following mix- 
ture of reagent grade chemicals well mixed is often uBed in the amount of 5 mg. /ml. of 
blood. 

Sodium fluoride, powder (NaF) 10 parts 

Thymol, powder , 1 part 

Potassium oxalate, powder 3 parts 

4. Citrate . Sodium and potassium citrates can be used as efficient anticoagulants, 
especially In the collection of blood for transfusion. For analytical purposes, citrate 
has the disadvantage of causing marked shifts of volume and acidity between erythro- 
cytes and plasma. About 60 mg. sodium citrate <Na 3 CgH 5 7 ' 2 (or 5) H 2 0) should be 
used for each 10 ml. of blood. For prothrombin times, some workers prefer the use 
of 3.8% sodium citrate instead of 2% potassium oxalate. 

5. EDTA - disodium salt. This is soluble to about 10%. It should be neutralized 
to pH 7. 4 before use by the addition of NaOH. The use of 10 mg. /10 ml. of blood suf- 
fices to prevent coagulation. For practical use 2 drops of the 10% solution may be 
used for each 10 ml. of blood. It is preferable to dry this solution In the collecting 
tube but if It is not dried, the error by dilution will be about 1% (0. 1 ml. in a total vol- 
ume of 10 ml.) The use of EDTA has some advantages in the determination of sedimen- 
tation rates, hematocrit, and platelet counting. The potassium salt Is fifteen times as 



73 



soluble as the sodium salt and thus may be more convenient to use. 
References to EDTA use: 

Am. J. Clin Path. 23, 613 (1953) 
" " " " 25, 1090 (1955) 
USAF Med. J. 4, 1556 (1953) 

6. Heparin . This substance is a naturally occurring substance found In liver and 
lung and is extremely effective In preventing the coagulation of blood. The very pure 
preparations require only 1 mg./lOO ml. The usual preparations are effective when 
used in the proportion of 0. 5 mg. /10 ml. of blood. Preparations are available as the 
sodium salt and as the calcium salt. Some preparations have significant amounts of 
phosphate as well. This Inorganic ion content should be considered when heparin Is 
used as an anticoagulant. 

Precautions: 



1. With all of these anti-coagulants, If plasma is to be used, it should preferably 
be separated from the rbc as quickly as possible. 

2. If whole blood is to be used, it is extremely important to thoroughly mix the 
sample of blood before a sample is withdrawn. If this is not done, any constituent with 
unequal distribution (by volume) in erythrocyte and plasma will be estimated with an 
original " built-in" error. This error, due to inadequate mixing, is very common in 
most clinical chemistry laboratories . It can be eliminated only by unending vigilance 
on the part of the director and the individual laboratory technicians. 

Changes In stored blood : 

Certain changes in the chemical constitution of blood, some of which are listed be- 
low, occur when blood is stored. In general, It is best to analyze samples as soon as 
possible after collection. If serum or plasma is to be used, prompt separation from 
the cell mass Is recommended. 

Some of the possible changes are: 

1. Loss of carbon dioxide: this Is more rapid when the exposure to the air Is in- 
creased. When this occurs, the cells are involved by way of the cliloride shift (see 

p. 34). To minimize this loss of carbon dioxide, blood may be collected without con- 
tact with air in "Vacutainer" tubes (see p. 75 ). under oil or under mercury, or merely 
kept in a capped syringe with suitable anticoagulant until it is analyzed. 

2. Glucose Is metabolized: the red blood cells utilize glucose and part at least is 
converted to lactic acid. This results in a low blood sugar and also in pH changes in 
the blood. 

3. Plasma inorganic phosphate may increase due to hydrolysis of sugar phosphate 
esters In the rbc and their transfer to the plasma. Prompt separation of plasma or 
serum avoids this. 

4. Ammonia may be formed by bacterial action on the urea of blood, and this pro- 
cess is quite rapid in contaminated specimens. This formation of ammonia interferes 
In the cobalt! -nitrite chemical method for potassium. 

5. Some substances diffuse from the rbc to plasma. Potassium will diffuse (a) 
from plasma to rbc at first, (b) later from rbc to plasma. If whole blood Is kept cold, 



74 






the cell potassium will diffuse to the plasma. Prompt separation of serum and cells 
prevents this type of error. 

After separation, the samples of serum or plasma may be stored In the refrig- 
erator or frozen. Details of the effects of various types of storage on the individual 
determinations will be mentioned specifically (when they are known) under each 
method, 

"Vacutalner" Tubes: 

It 1b possible to obtain special evacuated tubes, which can be prepared contain- 
ing anti-coagulant, from several commercial sources. These make it possible to 
collect venous blood without exposure to air and thus without loss of carbon dioxide. 



























75 



PROTEIN-FHKE FILTKATES 



< 



Introduction: 

In most of the determinations to follow, the removal of protein from the solution 
Is the first step in the analysis. Proteins remain in solution because the forces tending 
to separate the individual protein molecules are greater than the forces tending to bring 
them together. These separating forces are: 

1. Electrical charge on the protein. This Is present whenever the protein is away 
from its isoelectric point, i.e. , when It is Ionized . 

2. Hydration. Since most proteins contain many polar (hydrophllic) groups, a 
shell of water forms around the protein molecule. 

Any procedure tending to decrease the magnitude of these two separating forces 
will tend to precipitate the protein. 

Some general procedures used to precipitate proteins are as follows: 

1. Adjustment of pH to the Iso-electric point. Some proteins precipitate when 
brought to their iso-electric point. For example; the casein of milk will precipitate 
when the milk is "soured" or "brought to the iso-electric pH of casein. " The Iso-elec- 
tric pH (or point) Is the pH at which the number of negative charges on the protein par- 
ticle equals the number of positive charges; i.e. , there is a zero net charge. 

2. Alteration of hydration by the use of (a) strong salt solutions, (b) non-aqueous 
solvents (such as alcohol) at low temperatures and (c) evaporation of water at low tem- 
peratures (lyophllizatlon) all serve to precipitate proteins and usually result in little 
or no alteration in the structure or natural state of the protein; i.e. , little or no "de- 
naturation. " 

3. Denaturation of a protein produces a change (usually a decrease) insolubility. 
This denaturation may be brought about by (a) heat, (b) non-aqueous solvents at room 
or elevated temperatures, (c) strong acids or alkalies, and (d) other types of reagents 
or physical procedures. 

4. When proteins are charged (ionized) they will combine with other Ions to pro- 
duce insoluble compounds. 

(a) When they have a positive charge, - I.e. , they are cations (existing as proteinn 
they will combine with "heavy anions" (called alkaloidal reagents) such as picrate, fer- 
rocyanlde, tungstate, etc. ,' to give less ionization. 

(b) When they- have a negative charge, i.e. , they are anions (protein - ) they will 
combine with heavy metal ions or other heavy cations such as mercuric, lead, zinc, 
etc. , to give decreased ionization. 

With each of these methods using heavy cations or heavy anions, there is a more 
or less complete denaturation of the protein when the precipitation occurs at room 
temperature or above. If precipitation with heavy metals is carried out at low tem- 
peratures (0°C.) the protein may be recovered In essentially its native state. 



76 









There have been many different procedures used for the preparation of proteln- 
free filtrates. The most commonly used one Is that Involving tungstlc acid. Unless 
otherwise noted In the specific method, a protein-free filtrate refers to a filtrate 
prepared as noted In the tungstlc acid methods below, 

METHOD I 

Reference : 

Folin, O. , and Wu, H. , A system of blood analysis, J. Biol. Chem., 38, 81 (1919). 

Reagents : 

1. 10% sodium tungstate. Dissolve 100 g. reagentgra.de, carbonate free, sodium 
tungstate (Na 2 W0 4 *2H 2 0) In water and dilute to 1 liter, mix. This solution is stable 
Indefinitely. 

2. 2/3 N H 2 S0 4 . Weigh out 35 g. of concentrated sulfuric acid in a small tared 
beaker, dilute to 1 Titer with water and mix. Check by titration against standard alkali 
and adjust if necessary. 

Procedure ; 

To 1 volume of blood In an Erlenmeyer flask, add exactly 7 volumes of water, mix. 
Add 1 volume of 10% sodium tungBtate and mix. Add slowly and with shaking 1 volume 
of 2/3 N H 2 S0 4 . Stopper the flask securely and shake hard. Only a few bubbles should 
form and a hard metallic splash should be heard. Let stand 3 minutes. Pour slowly on 
to a dry filter and collect the filtrate. 

METHOD H A. 

Reference : 

Haden "Modification of Folin Wu protein-free filtrates:- J. Biol. Chem., 56, 469 (1923) 

Principle : 

In this modification the blood Is laked and proteins given a positive charge by the 
acid; I.e. , they become cations. Then, addition of the tungstate results in the forma- 
tion of protein-tungstates. This procedure employs fewer solutions and gives more 
filtrate than Method I. 

Reagents : 

1. 10% sodium tungstate, as in Method I. 

2. N/12 H 2 S0 4 . Add 2, 5 ml. of concentrated H 2 S0 4 to 1 liter of distilled water. 
Mix well and check by titration against 0, 1 N NaOH so that 20 ml. of N/12 H 2 SO 
requires 16. 7 ml. of 0. 1 N_NaOH for neutralization. 

Procedure : 

1. In a 250 ml. flask place 40 ml. of N/12 H 2 S0 4 . 

2. Add slowly, 5.0 ml. of blood, mixing well. Allow to stand 1 minute. 



77 



3. Add 5,0 ml. sodium tungstate 10%, stopper, shake vigorously; let atand 3 
minutes. 

4. Pour slowly on to a dry filter. 

" In these two procedures the protein precipitate Is or becomes a chocolate brown 
and the filtrate should be absolutely clear and colorless. It contains all blood con- 
stituents except protein, diluted 10 times. 

METHOD II B. 

Principle : 

Since it Is desirable to bind all the tungstlc acid, when serum or plasma is used, 
a modification must be made since less protein Is present. Some workers have recom- 
mended 50% of the amounts used for whole blood, but we have found, along with many 
others, that 70% gives somewhat better results. 

Reagents : 

1. Dilute 7 volumes of N/12 H 2 S °4 U P to 10 volumes with water. This dilution 
gives 0. 058 NH 2 S0 4 . 

2. Sodium tungstate 7%. Dilute 7 volumes of Na-jWO^HgO (10%) up to 10 vol- 
umes with water. 

Procedure ; 

To eight volumes of 0. 058 N HLSOj add 1 volume of serum or plasma. Mix. Add 
1 volume of 7% sodium tungstate and shake vigorously. Filter through filter paper, or 
centrifuge. 

METHOD m . 

Reference : 

Van Slyke, D.D. and Hawkins, D. , Pre-mixed Tungstic Acid, J. Biol. Chem. , 79, 
739 (1928). 

Principle : 

The Bodium tungstate and the sulfuric acid are mixed before the blood, serum or 
plasma, or cerebro-spinal fluid is added. The tungstic acid produced is unstable and 
should be freshly prepared weekly. This procedure Is especially recommended when 
a large number of filtrates are made daily. 

Reagents : 

Mix: 1 volume of 2/3 N.HoSC> 4 
7 volumes of distllleu HO 
1 volume 10% Na 2 W0 4 (sodium tungstate) 



or 



8 volumes N/12 H 2 S °4 Prepare fresh weekly. Small amounts of 
1 volume 10% Na„W0 4 precipitated tungstic acid do not interfere. 



78 









Procedural 

A. Wfcgjg Blood: Te » volume of pre -mixed tungstle acid reagent, add 1 volume 
of whole blood. Shake and filter. 

B. Serum and Plasma : To a mixture of S volumes of tungstlc acid reagent and 

4 volumes of distilled water add 1 volume of serum (or plasma) dropwise with contin- 
ual shaking. Shake vigorously and allow to stand for 10 minutes. Filter. 

C. Cerebro-splnal fluid : To a mixture of 2 volumes of tungstlc acid reagent 
and 2 volumes of distilled water add 1 volume of spinal fluid. This obviously results 
In a 1-5 dilution Instead of the usual 1-10, and this should be taken into consideration 
In the calculation of results. 

METHOD IV 

Zinc sulfate -barium hydroxide 

Reference : 

Somogyl, M. , J. Biol. Chem. , 160, 69 (1945). 

Principle : 

The reaction between zinc sulfate and barium hydroxide is complex, but In essence, 
results In the precipitation of two Insoluble products, zinc hydroxide and barium sul- 
fate, which bind and occlude the protein, removing It from solution. This procedure 
has the advantage of adding no extraneous ions to the solution. 

Reagents : 

1. Zinc sulfate reagent 5%. Dissolve 50.0 g, of ZnS0 4 - 6HgO (or 7H 2 0) in water. 
dilute up to 1000 ml. and mix. 

2. Barium hydroxide 0. 30 N. Dissolve 50 g. of Ba(OH) • SHgO and dilute up to 
1000 ml. of water. This solution must be balanced against the zinc sulfate as follows: 

Pipet Into a flask 25.00 ml. of the zinc sulfate solution. Add phenolphthaleln 
indicator and titrate dropwise with the Ba(OH) 2 solution, using vigorous stirring, to 
a definite permanent pink color. Adjust the concentration of the barium hydroxide or 
the zinc sulfate. Store the alkaline solution with protection from carbon dioxide of the 
air. 

3. Dilute reagents. Add four parts of water to one part of the reagent. 

Procedure A. (For a 1-10 filtrate) 

1. Add to 5.0 ml. distilled water, 1.0 ml. of sample (whole blood, serum or 
plasma). To this add 2. ml. of 0. 3 N Ba(OH) 2 . Mix and allow to stand 3 to 5 minutes. 

2. Add 2.0 ml. 6% ZnSQ. and shake vigorously. 

3. Filter through a dry filter paper Into a dry container (or centrifuge) to obtain 
a clear filtrate. 



79 



Procedure B . (For a 1-21 filtrate) 

1. Plpet 10 ml, of dilute barium hydroxide Into a small dry flask and add 1.0 ml. 
of blood, serum or plasma. Mix well and allow to stand 3-0 minutes. 

2. Add 10.00 ml. of dilute zinc sulfate reagent, Btopper and shake. 

3. Filter through dry filter paper Into a dry container or centrifuge. Notice that 
In this case the total volume Is 21 ml. and the dilution Is 1-21. 

Note 1. 

The use of barlum-zlnc deprotelnlzation has the advantage of somewhat greater 
speed and somewhat larger volumes of filtrate can be obtained, ft removes some of 
the nitrogenous constituents contributing to the non-glucose reducing substances and 
therefore blood glucose values closer to the actual glucose concentration are obtained. 
It also results in lower normal non-protein nitrogen values and clearly the filtrates 
cannot be used for the determination of uric acid or creatinine since these do not ap- 
pear in the filtrate . 

Note 2. 

Other volumes and concentrations can be used provided the same proportion of re- 
agents is used (2 ml. each of 696 ZnSO. and 0.3. N Ba(OH)„ for each ml. of blood). By 
this method a clear filtrate may be obtained regardless oi how small an amount of 
protein may be in the biological fluid. This is not true of the tungstlc acid filtrate. 
The mixture can also be centrifuged and an aliquot removed by plpet or by decantatlon. 

Note 3. 

Sodium hydroxide may be substituted for Ba(OH)n of the same normality, when 
whole blood Is used, but some protein is left In solution when serum or plasma is used, 
unless heat is also applied. 

Other methods of precipitation: 

The methods mentioned above, are those upon whose use Is based moat of our basic 
clinical values, together with the pathological variations characteristic In diseases 
states. In some cases, the use of tungstlc acid filtrates has been found unsatisfactory 
and other methods have been resorted to. Examples of these will In most cases be 
found in the manual associated with those methods. These are reagents such as zinc 
and copper hydroxides, trichloracetic acid, picric acid, etc. In addition there are 
numerous modifications of the tungstlc acid and the metallic hydroxide procedures, 
each with some particular advantage. Those outlined above will suffice for most pur- 
poses. 

METHOD V 

Micro-filtrates: 

In some cases, the amount of sample available may be limited, making it necessary 
to reduce the volume of the reagents proportionally. Whenever a "to contain" pipet is 
used, It should be remembered that a "rinse-out" technique is required. 



80 






( 



Procedure A. Micro-modification of Method H A 

Into a test tube, which can be centrifuged, Introduce 1, 60 ml, N/12 H 2 90 4< 
From a finger tip (or heel or ear lobe) prick, collect 0, 20 ml. of blood in a "to contain" 
plpet. Rinse back and forth In the 1. 69 ml. N/12 H.90. at least eight times and then 
add 0. 20 ml. 10% sodium tungstate. Mix well. (The acid and tungstate may be mixed 
prior to addition of the blood. ) Centrifuge and plpet 1. ml. of the supernatant for 
analysis. 

Procedure B . Micro-modification of Method HI A. 

Into a test tube, which can be centrifuged, add 1.8 ml. pre -mixed tungstlc add 
reagent. From a 0. 2 ml, "to contain" capillary plpet, introduce the sample of blood, 
rinsing back and forth at least 8 times. Centrifuge and plpet an aliquot for analysis. 

Procedure C . Micro -modification of Method IV B, 

To 2.0 ml. of 0, 08 N Ba(OH) 2 or (NaOH) add, using a 0. 2 ml. "to contain" plpet, 
0.2 ml. blood, rinsing as described above. Add 2.0 ml. 1% ZnSO^, mix well, centri- 
fuge and use the supernatant as a 1-21 diluted blood filtrate. 



§1 












( 



ACETONE (and ACETONE BODIES) la SERUM 



Reference b : 

(1) Dumm, R. M. , and Shipley, R. A., The Simple Estimation of Blood 
Ketones in Diabetic Acidosis. J. Lab. Clin. Med. 31, 116? (1946). 

(2) Greenberg, L. A., and Lester, D., A Micro-Method for the determin- 
ation of Acetone and Acetone Bodies. J. Biol. Chem. 154 , 177 (1944) 

(3) Shipley, R. A. , and Long, C.N.H., Studies on the Ketogenlc Activity 
of the Anterior Pituitary. Biochem. J. 32, 2242 (1938). 

Principle: 

Sodium nitroferricyanlde (sodium nitroprusside) In the presence of weak alkali 
(ammonia) will give a purple color with aceto-acetic acid and with acetone. 
B-hydroxybutyric acid does not give a positive test. The test applied to acetoacetic 
acid is five to ten times more sensitive than when applied to acetone. 

Reagents : 

Acetone Test Powder 

Grind separately (if not a fine powder) the following ingredients: 
Sodium nitroferricyanlde 1 gram 

Ammonium sulfate (dry) 20 grams 

Sodium carbonate (anhydrous) 20 grams. 
The three ingredients are mixed, without grinding, in a screw-capped 
bottle. The mixture must be kept dry at all times and under these conditions is 
stable for three months or longer. 

Procedure : 

1. For each test, place a pinch of the mixed powder, 5 millimeters in diameter, 
on a circle of white filter paper. Add one drop of serum without stirring. A positive 
test is indicated by a red to purple color. 

2. If a positive test Is obtained by undiluted serum, make successive dilutions 
by adding water to the sample, testing each dilution as above. 

This procedure may be applied to urine and to spinal fluid. 






( 

Calculations : 

The minimal concentration detectable under these conditions Is 10 mg. acetone 
per 100 ml. serum. Thus the dilution factor multiplied by 10 gives the approximate 
concentration of acetone bodies (expressed as acetone) present In the original serum. 

Example; 

A serum from a diabetic In coma was successively diluted and tested. 
The dilution of ( 1 / 6) gave a positive test and that of (1 / 6) gave a negative 
test. 

6<dilutlon factor) x 10 ■ 80 mg. % acetone 

Standardization: 

In order to check the reliability of the test, a known acetone solution of about 
B0 mg. % acetone may be prepared as follows: To 100 ml. of distilled water add 
2 drops pure acetone - mix well. Use as serum In the procedure outlined above. 

Interpretation : 

The normal serum contains 1-6 mg. of acetone bodies (as acetone), m diabetes 
elevations up to 300 - 400 mg. per 100 mi. may be seen. In normal urines up to 
50 mg. per day are seen while in diabetes 10 to SO grams per liter may be found - 
over half in the form of B -hydro xybutyrlc acid (which does not react In this test). 

This test is valuable in distinguishing between true diabetic acidosis In which 
the serum acetone exceeds 60 mg.% and surgical indications in a diabetic, e.g. , 
acute abdomen with vomiting, etc. . in which the blood level rarely exceeds 96 mg.%. 






( 



84 






ADRENAL INSUFFICIENCY iJSEl 
(Kepl.i -Tower Walti TeiA; 

References : 

(1) Robinson, F. J., Power, M. H. , and Kepler, E. J.i Proc, Mayo Clinic 
IS, 577 (1941) 

(2) Levy, M. 8., Power, M, H. , and Kepler, E, J.j J. Clin. Endocrinol, 
6, 607. (1946) 

(3) Cutler, H. H. , Power, M. H. , Wilder, R. M. : J. A.M. A. Ill, 11T (1938) 
Principle : 

Patients having Addison's disease show 

(1) a decreased ability to rapidly excrete water after Increased intake 

(2) excessive amounts of NaCl are excreted and there Is a tendency to 
retain urea. 

Procedure 1 : (Based on volume of urine) 

The Water TeBt - On the day before the test the patient eats three ordinary 
maals but omits extra salt. He is requested not to eat or drink anything after 6 
o'clock In the evening. Until tlds time he may drink water as desired. At 10:30 
P. M. he Is requested to empty his bladder and discard the urine. All urine which 
is voided from then on until ind Including 7:30 A, M. Is collected. The volume of 
this urine is measured and saved for chemical analysis If this should be necessary 
later. Breakfast Is omitted. The patient is asked to void again at 8:30 A.M. and 
Immediately thereafter he Is given 20 ml. of water per kilogram of body weight 
(9 ml. per pound). He is asked to drink this within the next forty -five minutes. 
At 9:30, 10:30, 11:30 A.M. and at 12:30 P.M. he Is requested to empty his bladder. 
in order to eliminate the effects of exercise and posture on urinary excretion, he 
Is kept at rest in bed except when up to void. Each specimen is kept In a separate 
container. The volume of the largest one of these four specimens is measured. 

Under these conditions some patients having Addison's disease will excrete so 
little urine that they are unable to void more than once or twice during the entire 
morning. In such Instances the am6unt of urine excreted per hour may be calculated; 
frequently however, such calculations are unnecessary because of the very low uri- 
nary output throughout the entire morning. 

Inferences that may be drawn from Procedure 1 : These are as follows: 

1. If the volume of any single hourly specimen voided during the morning Is 



85 



greater than the volume of urine voided during the night (10:30 to 7:30 A.M.), the 
response to the test is negative, that Is, such a response Indicates the absence of 
Addison's disease. The authors state that they have not encountered any exceptions 
to this rule. 

2. If the volume of the largest hourly specimen voided during the morning la 
less than the volume of urine voided during the night, the response to the test Is 
positive, that Is, Addison's disease may or may not be present. To establish the 
diagnosis, Procedure 2 should be Instituted. 

Proce d ure 2 : (Based on C hemistry of blood and urine ) 

To carry on with this procedure blood Is drawn, preferably under oil (or In a 
vacutalner tube), while the patient Is still fasting, and the plasma analyzed for Its 
content of urea and chloride. The specimen of urine which was voided during the 
night Is also analyzed for urea and chloride, From these four determinations and 
from the results obtained from Procedure 1 the following equation is solved: 



urea In urine (mg.%) CI* in plasma (mg. %) R volume of day urine (ml. \ 
urea in plasma (mg. %) C V in urine (mg. % volume of night urine (ml, ) 



The term "day urine" applied to the largest of the hourly specimens voided during 
the day; "night urine, " to the entire amount which was voided from 10i30 P.M. to 
7:30 A. M. It is Immaterial how these values are expressed provided that the same 
method be used throughout the equation. For example, if the concentration of plasma 
chloride is expressed as mg. of NaCl per 100 ml. the cone, of urinary chloride 
should be expressed in the same way. 

Inferences that may be drawn from Procedure 2 : These are as follows: 

1. If the value of A in this equation Is greater than 25, the patient probably does 
not have Addison's disease. 

2. If the value for this equation is less than 26, the patient probably has 
Addison's disease provided that renal failure has been excluded. 

The authors have encountered low ratios in cases of nephritis, diabetes Insipidus 
and in case of dehydration and fever. With the exception of two cases the question of 
Addison's disease did not arise. In these two Instances, the Cutler-Power-Wilder 
test excluded Addison's disease. Use of a more complex mathematical expression 
than the one given has also served to distinguish the response of patients having Addi- 
son's disease from those having nephritis. 



86 



( 









K the results of Procedure 2 are at all equivocal or If they are not Indicative of 
Addison's disease when there Is strong clinical evidence to the contrary, the test 
devised by Cutler, Power and Wilder may be Conducted, This can be instituted im- 
mediately. When this Is done, none of the patient's time Is wasted since the day of 
the "water test" constitutes the first day of the provocative test. Thus far we have 
encountered only two Instances in which it was necessary to resort to the Cutler- 
Power-Wilder test and in those cases it also yielded Indecisive results. 


















87 


















ALCOHOL, ETHVL 
(Blood, Urine, etc) 

Hefprences ; 

1. Bogen, E. : A quantitative study of acute alcoholic lntoxlatlon, Am. J. Med. 
Scl. U6, 153 (1928). 

2. Hall, W.W.: Drunkenness. Navy Medico -legal Aspects, U.S. Navy Med. Bull. 
34. 149 (1936). 

3. Newman, H. W. , and Ashenburg, N. J.: A quantitative study on intoxication. 
U.S. Navy Med. Bull. 44, 744 (1945). 

4. Oradwohl, Legal Medicine, Mosby Co. , (1954). 

Principle : 

The sample is aerated in the presence of a reagent which fixes certain non- 
alcoholic reducing agents. The volatilized alcohol is then drawn over into and reacts 
with potassium dichromate as follows: 

2K 2 Cr 2 7 / 8H 2 S0 4 / 3C 2 H 6 OH > 2K 2 S0 4 / 2Cr 2 <S0 4 )g / IIHjO / SCHgCOOH 

The reduction of Cr^ 6 — > Cr^ 3 involves a change of color from yellow to green. 
The degree of color change Is compared to a set of known standards. 

Apparatus : 

See illustration of aeration apparatus page 88a. 

Reagents : 

1. Anstie's Reagent : 

Prepare a 50% (V/V) solution of sulfuric acid In the usual manner (see 
stock reagents p. 20). Weigh out 3.333 g. reagent grade potassium dichromate 
(K„Cr 0_) dried 24 hours at 110°C. Dissolve the potassium dichromate in about 
900 mi. 50% H 2 SO in a liter volumetric flask and dilute to the mark with 60% H-SO,. 
This reagent is stable but keep it protected from direct sunlight. 

2. Scott-Wilson Reagent : 

Dissolve 1 g. mercuric cyanide, Hg(CN) 2 in 60 ml. distilled water. Add 
a solution of 18 g. of Sodium hydroxide NaOH in 60 ml. of distilled water, stirring 
vigorously. Add slowly with stirring 0. 29 g. silver nitrate dissolved in 40 ml. dis- 
tilled water. The mixture should be clear and may be used immediately. If turbid, 
allow to stand and decant the clear supernatant for use. This reagent keeps about 2 
months . 

3. Liquid Petrolatum (paraffin oil): The silicone defoamer, Dow-Corning Anti- 
foam A, may be used. 

Procedure : 

1 . Arrange the aeration apparatus consisting of a train of five tubes as shown in 
Fig. 21, p. 88a. 



88 



( 



l_ 









Air in 




Ansf ie's | ec Wood or urine 
reagent 3 cc. water 

I cc. Scott-Wilson 



V^ 



Empty tubes 



Anstie't 
reagent 




Aeration system 
tube holder 



Figure 21, Bogen's blood alcohol apparatus. 






88a 















( 






























< 


















2. Add to the tubes as follows: 

Tube 1-5 ml, Anstie's reagent 

2 - 1.0 ml. sample (Ostwald ptpet) 

1. ml. Scott-Wilson reagent 

4ml. HO 

5-10 drops liquid petrolatum or anti-foam 

3 - nothing added 

4 - nothing added 

5 - 6. 00 ml. Anstie's reagent (volumetric plpet) 

3. Connect all tubes in the train making sure they are appropriately placed. 

4. Connect the outlet of the 5th tube to a suction pump for aeration; control the 
speed of aeration to 2-4 bubbles per second. 

5. Immerse the apparatus Into a boiling water bath and aerate for 10 minutes 
keeping the water bath boiling. 

6. After aeration period is finished, remove and cool the 5th tube fa cold water. 
Transfer the contents to a clean, dry tube, identical to the standard tubes and dilate 
up to 5.6 ml. mark with distilled water. 

7. Compare the unknown with a known Bet of standards. Report as mg. alcohol 
per ml. whole blood. 

8. If the alcohol content is 4 mg. /ml. or greater repeat the test using 0. 6 ml. 
blood. Multiply the final result by a factor of 2. 

Standa rdizatlon: 



Prepare a 1. 0% solution of alcohol in water by adding 1. 27 ml. absolute alcohol 
at 15°C. (1.26 ml. at 20°C.) to 50 ml. distilled water in a 100 ml. volumetric flask. 
Dilute with distilled water to the mark. One ml. standard contains 10 mg. alcohol. 

Prepare eleven tubes labeled 0.0 to 5.0 mg. Add to each the desired volume of 
1% alcohol solution; e.g. , 0.05 ml.- to the 0.5 mg. tube - 0.5 ml. to the 5.0 mg. tube. 
To each tube add 5.00 ml. Anstie's reagent, rinsing the side of the tube during the 
addition. Add distilled water to each tube to bring the total volume to 6. 5 ml. Beat 
the tubes in a boiling water bath for 10 minutes, remove and cool. Draw and seal the 
ends of the test tubes in a flame. These standards should be stable for a period of 
3 to 4 years. 

a is essential for accuracy in this determination that extreme care be taken to use 
scrupulously clean glassware. Traces of reducing materials such aa soap, detergents, 
proteins, sugars, etc. will interfere in the test by reacting aa alcohol does. The Scott- 
Wilson reagent will unite with most ketones and prevent their interference naleee the 
level le very high. 

It ia important to avoid the use of alcohol, acetone aad ether In the akm prepara- 
tion for venipuncture in this determination. 

H. Is good practice occasionally to run a blank determination on distilled water to 
check the reagents. 



89 



( 









Other biological fluids may be used Instead of blood, such as urine, spinal fluid, 
and tissue. For spinal fluid analysis see Gettler and Frelreich: J. Biol. Chem. 92, 
199 (1930). For precautions In the analysis of tissues see Jetter and McLean: Am. 
J. Clin. Path. 13, 17B (1943) who state that the alcohol level In the body does not 
appreciably change in the 24-48 hours after death - at least In temperate zones. Actual 
post-mortem putrefaction may raise the level to as much as 5 mg. per ml. 

Interpretation : 

Normal persons may show a level In this test as high as 0.5 mg. per 1 ml. of 
whole blood. The level of brain alcohol lags somewhat behind that of the blood. The 
individual shows considerable variation in clinical response to a given level of blood 
alcohol as shown by the following table: 

Blood alcohol level of % of Individuals showing 

persons suspected of Intoxication symptoms of Intoxication 

0.6 10% 

1.0 10-18% 

1.6 22-47% 

2. 35-84% 

2.5 80-90% 

3.0 95-100% 

4.5 100% 

S Is an established fact that the intoxicating effect of alcohol like many other drugs 
is proportional to the amount present in the tissues. la this case the tissue most im- 
portant is the brain. As the concentration In the blood bears a direct relation to that in 
the brain, the examination of the blood offers an accurate index of the brain's alcoholic 
content. The highest blood concentration is usually reached about 1 hour after a given 
dose. Urine excreted from blood of a given concentration has a concentration equal to 
or slightly higher than that of the blood. If the concentration of alcohol in blood Is 
1.00, then urine would be 1.25, and brain 0. 90. The breath concentration is also 
proportional to that of the blood. 

It should be remembered that for Navy purposes, drunkenness is defined as "any 
intoxication sufficient to sensibly Impair the rational and full exercise of the mental 
and physical faculties. " Article 112, Manual for Courts Martial. 

It may be advisable, in critical medico-legal examinations, to run simultaneous 
Bogen's blood alcohol determinations on the patient and on the analyst, so as to elim- 
inate the possibility of contamination. 



90 









ALCOHOL - METHYL 
Serum 

Reference : 

Ostium, E.E.: U. 8. Naval Medical Bulletin 46, 1170 (1946) 

Principle: 

A protein-free filtrate of blood or other protein-free material Is treated with an 
oxidizing agent (at room temperature to differentiate from other substances such as 
ethyl alcohol) to convert methyl alcohol to formaldehyde. Chromotroplc acid (1, 8 
dlhydroxynaphthalene -3,6 dlsulfonlc acid) reacts with formaldehyde In the presence 
of sulfuric acid and heat to give a purple color. 

If chromotroplc acid Is not available a Leach test may be performed using milk - 
see below. This Is a somewhat less sensitive test. 

Reagents : 

1. 20% (w/v) Trichloroacetic acid. Dissolve 20 g. C CI. . COOH up to 100 ml. 
total volume. Mix well. Store In refrigerator. 

2. Acid potassium permanganate. To 3 g. KMnO . add 25 ml. phosphoric acid 
(H3PO . - 85% anal, reag.) , dilute up to 100 ml. Shake until the potassium perman 
ganate is dissolved. 

3. Sodium bisulfite (NaHSO ) solid, powdered anal, reagent. 

4. Chromotroplc acid (1, 8 - dlhydroxynaphthalene - 3, 6 dlsulfonlc acid) solid, 
powdered. Eastman Kodak* P230 '«"*dium salt). 

Procedure : 

1. Using a serological plpet, place 2 ml. of oxalated blood in a 26 x 200 mm. test 
tube. 

2. Add slowly jvith shaking 2 ml. of 20 per cent trichloroacetic acid. Stopper and 
shake well for 1 minute. 

3. Transfer to a centrifuge tube and centrifuge at 2,000 r.p.m. for 6 minutes. 

4. Plpet 1 ml. of the clear supernatant fluid into a test tube. (If the supernatant 
fluid is turbid, filter it through a small retentive filter paper.) 

5. Add 0. 2 ml. of the potassium permanganate reagent. Mix and let stand for 1 
minute. 

6. Add about 10 mg. of powdered sodium bisulfite to decolorize completely the 
solution of permanganate . 

7. To the water-clear solution, add a small amount of chromotroplc acid powder 
and shake. (Use approximately the amount of chromotroplc acid that will adhere to the 
end of a safety match.) 

3. Add 1.5 ml. of concentrated sulfuric acid, allowing the acid to flow down the 
side of the tilted tube to form a separate layer in the bottom of the tube. 

9. Examine the junction of the two layers against a white background. If methyl 
alcohol Is present, a purple ring will usually be noted. 

TO. Cautiously mix the contents of the tube by gently twirling, and allow the tube 



91 



to stand until the mixture has reached room temperature. If methyl alcohol was 
present In the original fluid, a diffuse violet color will be noted. 

11. It is advisable to perform a positive test by adding a drop of methyl alcohol 
to*2 cc. of normal blood and complete the procedure. Furthermore, a negative con- 
trol of the unknown should be run by omitting the chromotroplc acid in step T and 
continuing the procedure. Suspected beverages may be tested for methyl alcohol by 
the same procedure as was used for blood or body fluids. 

12. hi order to be sure that pre-formed formaldehyde (or a precursor such as 
methenamlne, a drug which liberates formaldehyde) Is not present, a test should 
also be run omitting the oxidation (Steps 5 and 6) . 

13. If chromotroplc acid is not available, the Leach test may be run as follows: 
To 10 ml. of milk (fresh, skim, or reconstituted), add 1 ml. of the filtrate 

obtained in Step 6 above. Add 10 ml. of concentrated (37%) HC1 containing 0.02% 
FeCl„. Heat to boiling. A violet color Indicates the presence of formaldehyde. The 
positive and negative controls indicated in Steps 11 and 12 should also be run with 
this test. 

Calculation: 

This is a qualitative test. A rough Beml-quantitation may be made by adding var- 
ious known amounts of methyl alcohol to blood and comparing the unknown with these. 

Interpretation : 

The actual level of methyl alcohol has little relationship to clinical toxicity. 
Methyl alcohol is metabolized in the body to formic acid (H - COOH) which is the 
actual toxic agent. One of the very serious effects of formic acid toxicity is its 
effect on the optic nerve resulting in blindness which is more or less complete and 
may be permanent. 

92 



AMINO- AC IDS 
In Plasma and Urine 



References : 

Shroeder, W.A. , Kay, L.M., and Mills, R.D.: Anal. Cbem. 22, 760 (I960). 

Albanese, A. A. , and Irby, V.: J. Lab. Clin. Med. 30, 718 (1945). 

Albanese, A. A. , and Irby, V. • J. Biol. Chem. 153, 683 (1944). 

Fister, H.J. , Manual of Standardized Procedures, Standard Scientific Supply 
Co. (1960) 

Principle: 

Amino-acidB in neutral solution react with alkaline copper phosphate to form soluble 
cuprlc salts usually of the form A„Cu. After the excess copper phosphate is removed 
the soluble copper in the filtrate is determined by lodimetric titration. Ammonia, does 
not contribute to the copper value nor does creatine, creatinine, uric acid or urea. 
However, dipeptides, tripeptides and In general the "end-group" amino acids with a 
free carboxyl adjacent to a peptide -linked nitrogen or with a free amino group adja- 
cent to a peptlde-linked carboxyl group. It has been shown to include urine citrate also. 

Apparatus : 

Micro-burets 

10 ml. (or less) for urine 
4 ml. (or less) for plasma 

Reagents : 

1. Cupric chloride - 28 g. of reagent grade CuClo^BLO per liter of water 
solution. 

2. Sodium phosphate - 68.5 g. of reagent grade Na„PO . 12 H„0 per liter 
of water solution. 

3. Sodium tetra-borate buffer - pH 9. 1 to 9.2 . 40. 3 g. of reagent grade anhy- 
drous Na„B O- (or 76. 4 Na B.Cy 10H 2 O) are dissolved in 4 liters of water and filtered. 

4. Washed copper phosphate - To 40 ml. of sodium phosphate solution is added 
20 ml . of cupric chloride solution with swirling. The suspension is now centrifuged 
for 5 minutes. The precipitated copper phosphate is washed twice by resuspension In 
60 ml. of sodium tetra-borate solution followed by recentrifuglng. The washed pre- 
cipitate is suspended in 100 ml. sodium tetraborate solution and 6 g. of reagent grade 
sodium chloride (NaCl) is dissolved in the suspension. The suspension is stable in 
glass-stoppered flasks up to 4 to 10 days. 

5. Thymolphthaleln Indicator - 0. 25 g. of thymolphthalein Indicator Is dissolved 
in 75 ml. absolute methyl alcohol (or 50 ml. 95% ethyl alcohol) and diluted to 100 ml. 
with water. 

6. Sodium thiosulfate 0.1N - Dissolve 25 g. Na„S Og-SHgO in 200 ml. distilled, 
boiled, and cooled water. Add and dissolve 11. 4 g. oorax Na„B 0_* 10H 2 O (or 6.0 g. 
anhydrous Na„B 0„) and 15 ml. iso-amy 1 -alcohol (the latter requires considerable 
water to dissolve). Dilute to one liter and mix well. 



93 






1. Futaefilum lodate standard - Dissolve 0.3S6T g. dry KIO- tip to 1 liter with water. 
Use to standardize the 0.0 IN and 0.00 IN sodium thiosulfate solutions (using 10.00 ml. 
and 1.00 ml. respectively). 

'8, Starch Indicator - Dissolve 1 g. Llntner soluble indicator starch In 100 ml. Of 
saturated NaCl by heating on the steam bath, cool overnight, and decant the supernatant 
solution. 

9. Potassium Iodide - Prepare just before use. 10 g, analytical reagent KI dis- 
solved up to 10 ml. with water. 

10. Glacial acetic acid - analytical reagent. 

11. NaOH. 1N_. 40 g. diluted up to one liter with water. 

12. Trichloroacetic acid 10"$ w/v 

Procedure : 

Urine 

Preservation - 24-hour specimens are collected In brown bottles containing 
00 ml. KCl (dilute IS ml. anal. reag. cone. HCl up to 100 ml. with water) and 1 ml. of 
10% alcoholic thymol solution . The total volume is made up to 2, 000 ml. and mixed well 
before removal of samples. Amino acids In urine are stable up to one week at room 
temperature. 

Method - To 15 ml. of urine sample In a 50 ml. volumetric flask are added 4 
drops of thymolphthalein indicator and 1TJ NaOH to a faint green or blue color. Then 
30 ml. of copper phosphate suspension are added from a graduated cylinder and the 
volume Is brought to the mark with distilled water. Mix thoroughly by repeated, com- 
plete inversion and allow to stand for five minutes. Filter through No. 5 Whatman 
(similar) paper filters into 125 ml. Erlenmeyer flasks. 

The copper content of 10. 00 ml. allquots of the filtrate is determined as follows? 
Each aliquot is acidified by 0. 6 ml. glacial acetic acid. To this acidified solution Is 
added 1 ml. KI solution. The solutions are then titrated with standardized 0.01 j^ 
sodium thiosulfate from a 10 ml. micro-burette. Six drops of starch indicator are 
added when the solution is a very light yellow and the mixture is titrated to a colorless 
end-point. 

Calculation ! 

Since two moles, of amino nitrogen combine with one copper atom, each ml. of 
0.0 IN thiosulfate is equivalent to 0.28 rag, amino nitrogen. Assuming exactly 0.01JJ 
thiosulfate was used to titrate 10. 00 ml. of filtrate then - 

ml. thiosulfate x P - i i ffi. = mg . amino N/ml. of sample 
3 

Example : A 24-hour collection of urine was brought to a total volume of 2000 ml. 
and treated as above. Two 10. 00. ml. allquots were titrated and a standardization was 
carried out and the following data collected. 



94 



( 









9.79 ml. of approximately 0.01 N sodium thiosulfate was required to titrate 
10.00 ml. of 0.01 N standard potassium iodate. 

N of thiosulfate = 10 - Q0 x 0.01 B 0.01021 N 
*" 9.79 ~ 

f - 1.021 

Two 10.00 ml. filtrate allquots (each representing 3.00 ml. of original sample) 
required 2. 10 and 2. 12 ml. for titration: 

amino N/ml. original sample - 2.11 x 1.021 x 0.28 

3 
= 0.2015 

amino N/24 hours = 0.2015 x 2000 = 403.0 mg. 



Plasma or Serum 






Method - In a 15 ml. centrifuge tube place 3.00 ml. 10% trichloroacetic acid, 
Add drop wise with mixing 1.00 ml. of plasma or serum. Mix thoroughly and allow to 
stand for 10 minutes, then centrifuge for 5 minutes. The supernatant solutions are 
decanted through Whatman #5 filter paper into test tubes and 2.00 ml. allquots removed 
to graduated (at 10 ml.) 15 ml. conical centrifuge tubes. 

To each tube is added in succession one drop of thymolphthalein Indicator, NaOH, 
1 N, to the appearance of a blue color, 5 ml. of cupric phosphate suspension and dis- 
tilled water to the 10 ml. graduation. 

After being mixed by vigorous shaking, the reaction mixtures are allowed to 
stand for 5 minutes and are then centrif uged for 5 minutes. 5.00 ml. allquots of the 
supernatant are pipetted into 125 ml. Erlenmeyer flasks. They are acidified with S ml. 
glacial acetic acid. Then 1.0 ml. of Kl solution and 3 drops of starch indicator solution 
are added and the sample titrated with standardized 0. 001 K sodium thiosulfate. from a 
very fine tipped 5 ml. microburette to the disappearance of the blue color. 

Calculations : 

Each ml. of 0.001 N thiosulfate is equivalent to 0.028 mg. amino N. Therefore 

since the final sample represents 0.25 ml. serum: 

mg. amino N per 100 ml. serum s ml. thiosulfate x factor x 0.028 x MP- 

0.25 

Example : In the standardization of the 0.001 N sodium thiosulfate 9.42 ml. were 

required to titrate 1.00 ml. of 0.oi'N.KIO„. (The factor therefore was 10 f 9.42 = 1.062.) 

5 ml. of final filtrate obtained as directed above required 0. 65 ml. of this sodium 

thiosulfate for titration, therefore: mg% amino N .-: 0.65 x 1.062 x 0.028 xiPP- =7.74 

0.25 



95 









Standardization 

0. 01 N and 0. 001 N Na 2 S 2 3 are prepared from 0. 1 N (see stock reagents) by 
dilution with distilled water using volumetric plpets and flasks. 

0.01 NNa 2 S 2 3 

To 10 ml. of 0.01 N KIO, , add about 4 ml. of water, 0.6 ml. glacial acetic acid, 
1 ml. KI solution, and titrate with 0.01 N Na 2 S 2 0„ to a very faint yellow. Add six drops 
of starch indicator and continue the titration to a complete disappearance of the blue 
color. Calculate a factor. See p. 30. 

0.001 NNa 2 S 2 3 

To 1.00 ml. of 0.01 N KI0 3 add about 4 ml. of water, 0.5 ml. glacial acetic acid, 
1 ml. KI solution, and 3 drops of starch Indicator. Titrate with 0.001 N Na^S.O, to 
the complete disappearance of the blue color. Calculate a factor. See p. 30. 

Notes : 

The analysis on serum or plasma must be carried out promptly. Hemolysis re- 
sults in markedly elevated values. Fresh urine specimens must be used or they may 
be preserved as outlined above. 

Interpretation : 

Urine amino nitrogen values by this method range from 200 to 700 mg. per 24 
hour period which is 3-4 per cent of the total nitrogen content of the urine. 

Increases in urine amino acids are seen in (1) acute severe hepatic Injury; (cyst- 
tinurla (a congenital renal tubular defect Involving lysine and arginlne); (3) Fanconl 
syndrome in which renal glycosuria and renal amino aciduria are both present; (4) Wil- 
son's disease which is hepatolenticular degeneration associated with a defect In copper 
metabolism. 

It is important to determine the amino N of urine in terms of per cent of total 
nitrogen. Normally between 3-4 per cent by this method - It may rise to 16 per cent 
in a Fanconl syndrome. 

Plasma levels are a little lower than serum levels, apparently because of reactive 
peptides released from fibrinogen during coagulation, and range between 4-8 mg.%. 

In severe liver damage, values as high as 200 mg.% have been reported. A de- 
crease is seen after the injection of insulin. 

For specific identification of amino acids, paper chromatography is used. See 
Dent, C.E. and Shilling, J. A.: Biochemistry Journal, 44, 318 (1948). 



( 









< 



96 



AMYLASE 
Serum, Plasma, Etc, 

References : 

(1) Somogyi, M. ; The Estimation of Diastase . J. Biol. Chem. , 125 . 399(1938). 

(2) Somogyi, M. : A New Sugar Reagent. J. Biol. Chem. , 160, 66 (1945). 

(3) Nelson, N. : J. Biol. Chem., 153, 375 (1944). 

(4) Smith, B.W. and Roe, J.H.: J. Biol. Chem., 179, 53 (1949). 

(5) Peralta, O. and Relnhold, J. G. : Clinical Chem. 1, 157 (1955) . 

Principle: 

The amylase is allowed to act on the substrate (cornstarch) for a period of time 
after which the reaction is stopped. The extent of hydrolysis Is then measured by 
determining the increase in reducing substances (sugars). The reducing substances 
may be determined by titrimetric (Ref. 1) methods or colorimetric procedures 
(Ref. 2 and 3). Alternatively, the decrease in substrate (starch) concentration may 
be measured by the decrease in iodine-starch complex color (Ref. 4) or In turbidity 
of the starch solution (Ref. 5). Only the colorimetric method will be described here. 

The maltose (reducing sugar) , liberated by the action of amylase on the starch 
substrate, is measured by the blood glucose method (p. 181). Because maltose is less 
active as a reducing agent, the period of heating with the alkaline copper reagent Is 
increased to twenty minutes instead of ten. Because of the presence of glucose and 
other reducing substances in the serum-starch mixture before incubation, a zero- 
time blank is necessary. After reduction, color development, measurement and 
calculations are carried out as for glucose In blood (p. 181). 

Apparatus : 

Colorimeter or photometer (500-550 kui). Folln-Wu (or similar) sugar tubes 
25 ml. 

R eagents : 

1. Starch paste . Grind thoroughly in a mortar 3 grams of washed starch with 
10 ml. of water and pour into 180 ml. of boiling water. Rinse the mortar with 10 ml. 
more of water. Boll one minute with agitation. Then cover the mouth of the flask 
with a beaker and set in a boiling water bath for 15 to 30 minutes. Then cool. Store 
under refrigeration. 

97 



%, Acid NaCl . 10 grams NaCl Is added to 3 ml. 0. 1 N HC1, dissolved In 
500 ml. water and diluted to one liter. 

3. Protein preclpltants 

a. 5%CuS0 4 -5H20 

b. 6% sodium tungstate 

4. Alkaline copper reagent 

See under blood glucose (p. 181). 

5. Arseno-molybdate (Nelson color reagent) 
See under blood glucose (p. 181). 



Procedure: 



1, For zero-time control 

a. To a 16 x 120 mm. pyrex test tube add exactly 6.0 ml. staith paste, 
2.0 ml. acid NaCl and 1.0 ml. 5% copper sulfate. 

b. Add 1.00 ml. serum and mix. 

c. Add 1.0 ml. 6% sodium tungstate solution, mix well, centrifuge and/or 
filter through a quantitative filter paper. 

2. For enzyme activity test 

a. To a 16 x 120 mm. pyrex test tube add exactly 6.0 ml. starch paste, 
2.0 ml. acld-NaCI and warm to 40°C. , then add 1.00 ml. serum, mix. 

b. Incubate exactly 30 minutes at 40°C. 

c. Immediately add 1.0 ml. 5% CuSO. and 1.0 ml. 6% sodium tungstate, 
mixing after each addition. Continue as for control tube in preparing 
a filtrate. 









98 









Colorlmetrlc Procedures: 
Introduce Into: 

1. Sugar tube C - 1.0 ml. control filtrate 

2. Sugar tube E - 1.0 ml. enzyme filtrate 

3. Sugar tube B - 1.0 ml. distilled water (reagent blank) 

4. Sugar tube S_ - 1.0 ml. 20 mg. % glucose standard (nominal 200 mg. % standard) 

To each of the four tubes add 1. ml. alkaline copper reagent and mix well. 
Heat in a boiling water bath 20 minutes. Remove and cool in water bath. Add 1.0 ml. 
color reagent, mix well until all cuprous oxide is dissolved. Dilute to 25ml. mark. 
Mix well by inversion. Compare photometrically (51S mu) as in the blood glucose 
method (p. 182) setting the reagent blank (B) at 100% T (D =. O). 

Calculation : 

Amylase activity is expressed as mg. glucose equivalents liberated In 1/2 hour 
per 100 ml. serum. Since the control tube also shows some reduction: 

E - C = amylase activity expressed as glucose equivalents (Somogyi units) 

The term glucose equivalents Is used to- denote the fact that whereas maltose 
Is the actual product of amy k> lytic activity, glucose Is the standard against which 
it is determined. 
► 

Example : Tube C - gave a reading of 63.0% transmlttance 
Tube E - gave a reading of 31. 4% transmlttance 
Tube B - was set at 100% transmlttance 
Tube S - gave a reading of 25. 0% transmlttance 

corresponding to the following optical densities 

C -0.201 
E - 0. 603 
B- 0.000 
S -0.602 

Using the usual photometric formula 

Cu = <Du/D 8 ) C 8 x ~ ree Glucose -blood (p. 182). 



C c = 66.6 
Cg = 167 
Then E - C = 100 mg. % - 100 8omogyi units 



Notes : 

On fluids with high activity. It Is more accurate to make a dilution with 
0. 85% NaCl before Incubation rather than after. 

Interpretation : 

Normal range - 40-120 Somogyi units Is the usual range although apparently 
some normals range up to 200 units. 

Significance - 

Elevations - are seen In acute pancreatic disease - less marked 
elevations are seen In chronic disease of pancreas. 

Decreases - are seen in liver disease. 

Acute pancreatitis - elevations may exceed 1000 units and may fall rapidly 
to normal values In 3-4 days. 

Chronic pancreatitis - Increases over base line values of 100-200 units. 

Other abdominal diseases such as: 

(1) Intestinal obstruction 

(2) Acute peritonitis 

(3) Perforated peptic ulcer 

hi these, smaller Increases not more than 600 units are seen developing 
5-10 days after the onset of the symptoms and appear to be due to Increased pressure 
on the pancreatic duct. 

Increases are also seen in mumps and obstructive disease of the salivary 
glands. 



100 






AMYLASE 
Serum 

Reference : 

Gomorl, G.,Am. J. Clin. Path. 27, 714 (1957) 

Hugglns, C, and Russell, P.S. , Ann. Surg., 128 , 668 (1948) 

Smith. B. W. , and Roe. J.H. , J. Biol. Chem. , 227, 357 (1957) 

Principle: 

A starch substrate Is acted on by the amylase In serum. The decrease In the blue 
starch-Iodine color Is measured photometrically. 

Reagents : 

1. Starch substrate. Dissolve 8 g. of Lintuer's soluble starch (Merck) and 1 g. 
benzoic acid In 800 ml. of boiling distilled water. Cool. Dilute up to 1000 mj. with 
distilled water. Mix well. This solution may be kept at room temperature and should 
be stable at least six months. 

2. 5% sulfuric acid. Dilute about 27 ml. of concentrated sulfuric acid up to one 
liter with distilled water. Cool. 

3. Iodine solution. Dissolve 1 g, of L, and 2 g. of KI in 50 ml. of distilled water. 
Dilute up to 300 ml. with distilled water. 

4. Phosphate buffer 0. 1 M pH 7. 4 

NaH 2 P0 4 *H„0 2.30 g.) Dissolve, dilute to one liter and mix. 

Na 2 HP0 4 anhyd. 11. 83 g. ) Check pH with glass electrode and standard buffer. 

Procedure: 



Into 3 large tubes calibrated at 60 ml. 
solutions and proceed as Indicated. 


, (T-test; 


C-control;and B-blan 


Solutions 


Tubes 


ja 


C T 


Starch substrate 

Buffer 

Serum 




— 


0.5 0.5 
0.5 O.S 
0.1 




Mix and Incubate at 37°C. for 
30 minutes. 


Sulfuric acid 5% 
Iodine solution 




2 

1 


2 a 
1 1 




Mix well, dilute to 50 ml. 
with distilled water. 



Compare photometrically at 640 mu using the blank to set at 100% T. 



101 






Standardization: 

1. Prepare a dilution of the starch substrate by diluting 10 ml. to 100 ml. with 
distilled water. 

2. Into 50 ml. volumetric flasks plpet diluted starch as follows: 



( 



Flask # 


ml. diluted 
0.0 


starch 


%of control 



m K 


. starch 


1 





2 


0.5 




10 




0.4 


3 


1.0 




20 




0.8 


4 


2.0 




40 




1.6 


5 


3.0 




60 




2.4 


6 


4.0 




80 




3.2 


7 


5.0 




100 




4.0 






3. Add 2 ml. of sulfuric acid 5% and 1 ml. 'of Iodine solution. Mix. Dilute to 
the mark and mix well. 

4. Compare photometrically at 640 mu using the blank to set at 100% T. 

5. Plot the O.D. vs. mg. starch and determine the optical density due to 1 mg. 
of starch. 

Calculations : 

D control ~ Ptest 
D l mg. 



x 100 - amylase units. / 



Note : 

If the starch Is completely decolorized or if a value of more than 400 units is 
found, the test should be repeated with a dilution of serum. Although the control contains 
4 mg of starch, this amount may be tripled and subsequently diluted to the factor. 



■ 

102 






ASCORBIC ACID 
Blood, Urine and Tissue 
Reference : 

(1) Roe, J. H. and Kuether, C. A. , J. Biol. Chem. , 147, 399 (1943) 

(2) Shaffert, R.R. , and Klngsley, Q.R. , J. Biol. Chem., 212, 59 (1965) 

Principle ; 

Ascorbic acid in blood or urine filtrate Is oxidized by means of activated charcoal 
(Norit) to dehydroascorblc acid (reversible oxidized ascorbic acid) which is then re- 
acted with 2, 4-dinitrophenylhydrazine to form a hydrazone. (Thiourea is added to pre- 
vent interference by oxidizing substances such as Fe++ + or HgGv which produce a color 
with 2, 4-dinltrophenylhydrazine.) The hydrazone derivative is now treated with strong 
sulfuric acid to produce a reddish color which is measured photometrically at a wave- 
length of 540 millimicrons. 

This procedure measures total ascorbic acid (reduced ascorbic acid plus dehydro- 
ascorbic acid) which may be partitioned by running two parallel determinations and 
omitting the activated charcoal treatment in one of them. Only dehydroascorblc acid is 
measured when this step is omitted. 

Reagents : 

Stock ascorbic acid standard - Accurately weigh exactly 100 mg. of L-ascorbic 
acid and place In a 100 ml. volumetric flask. Dilute to volume with 4 per cent tri- 
chloroacetic acid solution. 

Working ascorbic acid standard - Dilute 2 ml. of stock standard to 100 ml. with 
4 per cent trichloroacetic acid. 1 ml. = 20 micrograms of L-ascorbic acid. 

Trichloroacetic acid - 4 per cent solution and 6 per cent solution. (W/V). 

2. 4-Dlnltrophenylhydrazine - 2 g. of 2, 4-dinitrophenylhydrazine (Eastman) are 
dissolved In 100 ml. of 9 N Sulfuric acid. Let stand overnight and filter through What- 
man No. 42 filter paper. 

9 N sulfuric acid - Prepare by adding one part of concentrated HgSO . to 3 parts 
of water (V/V). 

85 per cent sulfuric acid - To 100 ml. of water add 900 ml. of concentrated sul- 
furic acid. 

Thiourea - Dissolve 10 g. of thiourea (Eastman) in 55 ml. of 95% ethyl alcohol and 

103 






dilute to 100 ml. with distilled water. This reagent should keep 2 mouths. 

Norit (activated charcoal) - Use U.S. P. grade. If the blanks read too high, add 
1 liter of 10 per cent hydrochloric acid (1 part concentrated UC1 to 9 parts water) to 
200 g. of Norlt In a large flask, bring to a boll, and filter with suction. Stir the cake 
with 1 liter of water and again filter. Dry the cake In an oven overnight at 100-120°C. 
Repeat If necessary. 

Oxalic acid -0.5 per cent (W/V) solution. 

Procedure : 

Standardization: Shake vigorously 25 ml. of working standard L-ascorbic acid 
with 1/2 teaspoon (0. 7 g. > of Norlt for 1 minute. Filter through Whatman No. 42 
filter paper. Add 0.0, 0.25, 0.50, 1.0, 1.6, 2.0, 2.5, and 3.0 ml. of filtrate to 
photometric cuvets and dilute each to 4 ml. with 4 per cent trichloroacetic acid which 
has also been shaken with Norlt and filtered. Add 1 drop of thiourea solution and 1 ml. 
of 2, 4-dinftrophenylhydrazine solution. Continue as under Development of Color . 
Step 2. 

Preparation of Filtrate: 

Whole blood and serum: 

(1) To 15 ml. of 6 per cent trichloracetic acid in a 50 ml. Erlenmeyer flask, 

add dropwise 5.00 ml. of sample mixing continuously. 

(2) Let stand for 5 minutes and centrifuge for 10 minutes at 2500 rpm. 

(3) Pour off the supernatant Into a clean, dry test tube, add 1/2 teaspoon of 
Norlt, and shake vigorously for 1 minute. Filter through Whatman No. 42 
filter paper. 



Urine: 






<1) Add 1 ml. of urine to 19 ml. of 4 per cent trichloroacetic acid. Add 1/2 
teaspoon of Norlt and shake vigorously for 1 minute. 

(2) Filter through Whatman No. 42 filter paper. 

Development of Color: 

1. To 4 ml. of filtrate In a photometric cuvet, add 1 drop of thiourea solution and 
1 ml. of 2, 4-dlnltrophenylhydrazlne solution. 

2. Place the tube In a boiling water bath exactly 5 minutes for blood or serum and 
10 minutes for urine and standards. At the end of this time place the tube r\ crushed 
ice for 5 minutes. 104 






3. Add slowly (drop by drop) 5 ml. of 85 per cent sulfuric acid, and mix by twirl- 
ing. Let stand for 10 minutes at room temperature. Read In the photometer (640 mu) 
against Its own blank, prepared in exactly the same manner, except for the omission of 
the 2,4-dlnltrophenylhydrazlne until after the addition of the 86 per cent sulfuric acid. 

Calculation 

Blood or serum : 

(D /D ) x C x (100/1) s ascorbic acid In mg./lOO ml. 

Using the 1.5 ml. standard (containing 30 ug. ascorbic acid): 

(D u /D ) x 0.03 x 100 = mg. % ascorbic acid. 

Urine : (Using the 1.5 ml. standard) 

(D u /D a ) x 0.03 x (1000/0.2) - mg. ascorbic acid per liter of urine. 

Example: A sample of whole blood treated as Indicated and read against Its own 
blank showed a transmlttance of 72 per cent. The 1.5 ml. standard (0.03 mg.) read 
against Its own blank, gave a transmlttance of 32 per cent. 

T (&) O.D. 

Unknown 72 . 143 

Standard 32 .495 

(0.143/0.495) x 0.03 x 100 = 0.87 mg. ascorbic acid per 100 ml. 

whole blood. 

Notes 

Reductones (degradation products of sugars) and dlketogulonlc acid (a product of 
lntra-molecular rearrangement of dehydroas corbie acid) as well as certain other al- 
dehydes and ketones, also, give hydrazones with 2,4-dinltrophenylhydrazine. The inter- 
ference is slight for all these except for diketo-gulonic acid because In the strong acid 
these products absorb only very slightly at the chosen wavelength (540 millimicrons). 
Diketo-gulonlc acid will be read as If it were dehydroascorblc acid. Its level in normal 
blood and urine samples Is close to zero. In tissues it may be a significant fraction of 
the total ascorbic acid content. For a complete fractionation of these compounds see 
Roe, J.H., et al. , J. Biol. Chem. , 174, 201 (1948), 

Interpretation 

The normal range for whole blood total ascorbic acid is 1. 2 to 2.3 mg. per 100 ml. 
In serum the normal range is 0. 5 to 1.4 mg. per 100 ml. Reduced ascorbic acid present 

105 



originally (omitting the Norit treatment) Is somewhat lower. 

la acute or recent ascorbic acid deprivation (2-3 weeks) tlie plasma or serum 
level may be close to zero but the whole blood levels will still be in the normal range. 
The white blood cells are the last to lose their ascorbic acid in deprivation studies. 



ASCORBIC ACID SATURATION TEST 

Principle: 

It is assumed that the tissues of a person with a previous history of insufficient 
ascorbic acid intake will take up quite large amounts when a test dose is given so that 
relatively little will be excreted in the urine. 

Various dosages , both oral and intravenous have been used, and various periods 
of urine collection have been chosen. It is recommended that the test be run in parallel 
with a person known to have a history of satisfactory ascorbic acid intake. 

Procedure : 

10 A.M. Give oralh/ 10 mg. ascorbic acid per kilogram body weight. 

2 P. M. Empty the bladder completely and discard the urine. 

4 P. M. Empty the bladder completely and determine the total ascorbic acid 
excreted in the two-hour period. 

Rppeat the test dally untii normal results are obtained. Ordinary 
meals may be consumed. 

interpretation: 

In normal persons an excretion of about 50 mg. (during the two-hour period) will 
be obtained on the first or second day. When a vitamin C deficiency Is present, a 
longer time, six to ten days, or up to several weeks may be required. 

The test has the obvious advantage of serving both as therapy and as a test. Pro- 
gress in therapy is indicated by the increased amount of ascorbic excreted as time 
goes on. 



106 



( 



< 












BILIUUUIK 
In So rum 

References : 

1. Malloy, H.T. , and Evelyn, K.A. , J. Biol. Chem. 119, 481-90 (1937). 

2. Ducci, H. r and Watson, C.J. , J. Lab. Clin. Med. 30, 293 (194S). 

3. Zicve, H1U, Hansom, Falcone, and Watson, J. Lab. Clin. Med, 38, 446 (1951). 

4. Altaian, V. , Nelson, M. , and Pernova, D. , Am. J. Clin. Path. 26, 956 (1956). 

Principle: 

Bilirubin, like aromatic amines, combines with diazonium salts to form azo dyes. 
In this case, sulfanilic acid is diazotized with nitrous acid (HCl / NaN0 2 ) and Is then 
coupled to bilirubin. The distinction between direct one-minute (l'B) ana total (TB) 
bilirubin depends upon the fact that addition of alcohol allows all the bilirubin to react 
with the diazo reagent and is used to make the reaction quantitative for total bilirubin 
content of the serum. Apparently there are two types of bilirubin: direct-reacting, 
polar, water soluble; and indirect-reacting, non-polar, alcohol soluble. 

Recently, Schmid has shown that the polar, water soluble, direct-reacting bili- 
rubin is bilirubin glucuronate which is formed by a conjugation process in the liver 
from free bilirubin which is the non-polar indirect-reacting type of bilirubin. 

Reagents : 

1. Methyl alcohol-absolute (or redistilled). 

2. Sulfanilic acid solution. Dissolve 1 g. of sulfanilic acid In 1 liter of dilute HCl 
(15 ml. concentrated HCl diluted to 1 Uter). 

3. Sodium nitrite solution: Dissolve 0. 5 g. NaNOg (ACS) in a little water, dilute 
to 100 ml. Prepare on day of use (or prepare from stock refrigerated 50% on day of 
use). 

4. Diazo reagent. Add . 3 ml. of the nitrite solution to 10 ml. of the sulfanilic 
acid solution. Mix. Prepare mixture within 5 minutes of time of use. 

5. Dilute HCl for blanks. Dilute 15 ml. of concentrated HCl to 1 liter with water. 

6. Bilirubin stock standard. Transfer exactly 20 mgm. of pure bilirubin to a 100 
ml. glass stoppered volumetric flask. Dissolve. Bring to mark with chloroform and 
mix. This solution is stable in the dark and cold, with well fitting stopper. 

7. Bilirubin working standard. Dilute 10 ml. of the above standard to 100 ml. with 
ethyl' alcohol 95%. This results in a concentration of 2 mg. /100 ml. 

Procedure : 

Collection of specimen : 

1. Post-absorptive to avoid lipemia. 

2. Plain tube (serum). 

3. Run test within 2 hours after clotting. After long standing only the indirect re- 
action occurs. 

4. Use filter or wavelength Indicated in calibration chart (530-560). 



107 



( 



Analysts of Scrum : (Note the necessity for step timing for direct bilirubin I) 

1. Pipette exactly 2.0 ml. of scrum into a clean dry test tube, add exactly 8.00 
ml. of distilled H 2 0, stopper and mix thoroughly. 

2. Pipette into each of two test-tube cuvets, a 4 ml. portion of this 1:5 dlluHoB of 
the serum. 

3. Add to one tube 1 ml. of dlazo blank solution, and to the other 1 ml. of the 
freshly prepared dlazo reagent, mixing well. 

4. After exactly one minute set the blank at 100. and read the unknown. 

5. Add 5. ml. of methyl alcohol absolute. Mix well, allow to stand 30 minutes 
for full color to develop. Read again as in step 4. 

Calibration curve : 

Prepare a calibration curve using 0.0, 1.0, 1.6, 2.0, 2.5, 3.0, 3.6, 4.0, 4.5, 
and 5. ml. of dilute working standard and ethyl alcohol to make 3.0 ml. Add 1.0 ml. 
diazo reagent, mix well. Head after 30 minutes. These standards correspond to up to 
10 mg, per 100 ml. for the direct and up to 20 mg. per 100 ml. for the total bilirubin. 

Calculation : 

1. Direct (l'B; bilirubin 

C u (mg.%) = (D u /D s ) x C 8 x (100/1) x (V u /V^ see page 57 
If <C S /Dg) x 100 = K and since V„ = 5 ml. and V 8 = 10 ml. 
then C u = D u x K x V u /V s ; 
C u = D u x (K/2) 

2. Total (TB) bilirubin (V u = 10 and V s a 10) 

C u = D u x K 

3. Micro-method (total bilirubin) seep. 109 

C u = (D u /D s ) x C 8 x (100/0.05) x (2.5/10) 



c u - D u x 5K 

Interpretation: 

Watson places the upper limit of normal l'B at 0.25 mg.% and the upper limit of 
TB at 1. 50 mg. %. An Increase mainly in the indirect reacting fraction indicates hemo- 
lytic jaundice, while an increase in the direct reacting indicates -an hepatic or post- 
hepatic cause of the jaundice.. 

The direct and indirect reacting bilirubin are not completely separated by this 
procedure. Only about 65% of the direct-reacting bilirubin is measured at one minute; 
practically all will be measured at 15 minutes. At this time however, some of the 
indirect-reacting bilirubin has started to react. Zieve et al, with Watson, found that 
the l'B gave the best clinical differentiation between the obstructive and the hemolytic 
types of jaundice. X08 









BILIRUBIN 
Micro-Method 

When frequent serum bilirubin determinations are required as tn neo-natal Jaundice 
due to Rh°(D> or other Incompatibility, it becomes desirable to be able to determine at 
least total bilirubin on amounts of blood obtainable by finger, heel, or ear puncture. 

Collection of Specimen ; 

See precautions as in macro-procedure. Make collection as outlined under Micro- 
collection (p. 70 ) obtaining 0. 3 to 0. 6 ml. of whole blood. 

Anah/sia of 8ernm : 

1. Pipet into a small test tube (B) exactly 0. 9 ml. distilled water. Add to this 
exactly 0. 1 ml. serum. 

2. Mix well and remove to another tube (A) 0. 6 ml. of the diluted serum. 

3. Add to tube (B) 0.6 ml. diazo blank solution and to tube (A) . 6 ml. diato 
reagent. 

4: Add 1. 5 ml. methyl alcohol to tubes (A) and (B) , mix well and read after 
16 minutes. 

Calculation: 

D x 6 K = mg-% total bilirubin. 

Caution: 

The use of the same raise for K for both macro- and micro- modifications of 
the bilirubin procedure assumes that the same size curat la used for standardization, 
and for reading the unknowns. 












109 



BROMIDE 
In Serum, Urine, and Spinal Fluid 



Reference b : 

1. Katzenelbogen, S. and Czarskl, T. , Proc, Soc. Exper. Biol, and Med. 32, 
136<1934). 

2. Dlethelm, O. , J. Nerv. and Mental Dis. 71, 161 (1930). 

3. For a more specific and accurate procedure see: Bloc hem. J. 66, 688 (1954). 

Principle : 

Bromide reacts with gold chloride to form gold bromide which gives a red color 
suitable for photometry. Chloride decreases the color formation. Sodium chloride Is 
incorporated into the reagents to correct for the effect of serum and cerebrospinal 
fluid chloride. 

Apparatus : 

Wavelength 440 mu faot critical) 

Reagents . 

Trichloracetic acid - chloride solution "TCA-NaCl. " 

Place 0. 6 g. reagent grade sodium chloride into a 100 ml. volumetric flask, 
dissolve in about 10 ml. distilled water. Add 10 g. crystalline trichloroacetic acid, 
dissolve and dilute to the mark. 

Gold chloride 0.5% 

Dissolve the contents of one ampoule (15 grains AuClg- HC1- 3IU0) In 196 ml. 
distilled water. Stir until dissolved and filter using an ashless filter paper. 

Sodium bromide - Stock Standard 

Place 1. 000 g. reagent grade sodium bromide in a 100 ml. volumetric flask. 
Dissolve and dilute to the mark with distilled water. 

Dilute working standard 

Transfer 3. 00 ml. of stock standard to a 100 ml. volumetric flask, dilute to 
the mark with distilled water and mix. Prepare immediately before use. 

Calibration : 

In a series of six test tubes, place 0.0, 1.0, 2.0, 3.0, 4.0, and 6.0 ml. of the 
dilute standard. Add water tc >nake 6.0 ml. These standards represent concentra- 
tions of 0, 30, 60, 90, 120, and 150 mg. /100 ml. of sample. Proceed immediately 
to the color development. 

To each standard tube add 4. ml. TCA-NaCl solution, and mix. Then add 1.0 ml. 
0.5% gold chloride solution and mix well. 

Within in minutes read each solution against the blank set at T = 100 or D - O at 



110 



( 









a wavelength of 440 mu. Plot the results which should fall on a straight line. Prepare 
a calibration table. 

Procedure : 

Serum; In a tube which can be centrlfuged place 8.0 ml. TCA-NaCl. Add drop- 
wise with constant shaking 2.0 ml. serum. Shake vigorously and allow to stand 30 
minutes. Centrifuge or filter through a small Whatman #2 or #44 paper. 

In a cuvet (sample) place 6.0 ml. clear filtrate, 4.0 ml. distilled water and 
1.0 ml. 0.6% gold chloride. Mix well. 

In another cuvet (blank) place 4 ml. TCA-NaCl, 6 ml. distilled water and add 
1.0 ml. 0.6% gold chloride and mix. 

Within 10 minutes read sample against the blank set at T - 100 (or D a O) at a 
wavelength of 440 mu. Obtain the concentration of bromide from the calibration table. 

Urine ; Proceed as under blood using a dilution of well-mixed mine. 

Spinal fluid ; Proceed as for serum. 

Interpretation : 

There is normally only a very small amount of bromide in serum and some of this 
value is undoubtedly due to non-specific color developed by serum filtrate. The normal 
low value Is usually said to be about 2-3 mg. per 100 ml. of serum. 

Symptoms of bromide intoxication appear when the serum bromide level reaches 
100 to 200 mg. % although there Is considerable Individual variability. Bromide partial- 
ly replaces chloride in the tissue electrolyte pattern and therapy consists in replace- 
ment of bromide by chloride. This may require several days to several weeks. 












Ill 



( 



BROMSULFALE1N TOLERANCE AND CLEARANCE TEBT 
(Sulfobromophthaleln) 

References 

1. Rosenthal, S. M. , andE.C. White; J. Am. Med. Assoc. 84, 1112 (1926). 

2. Gaebler, O.K.; Am. J. Clin. Path. 15, 462 (1945) 

3. Dragstedt, C.A. andM.A. Mills; J. Lab. Clin. Med., 21, 1306 (1936) 

4. Hamilton, R.H. ; Fed. Proc. 6, 258 (1947) 

5. Goodman, R.D. , and Kingsley, George R. ; J. A.M. A. , 153 . 462 (1953) 

Principles 

The dye , sulfobromophthaleln (Bromsulfaleln) is Injected Intravenously. S Is rap- 
Idly removed from the plasma by the normal liver; In liver disease larger amounts are 
retained In the circulating plasma after a given time Interval. 

The principle of the chemical determination Is based on the fact that the dye Is 
colorless in acid and reddish-violet in alkaline solution. This property Is utilized In 
the determination by measuring Its light absorption In an alkaline medium, m the 
presence of bilirubin, hemolysis or lipemla, the simple method described results In 
errors since the absorption characteristics of the Interfering substances are also al- 
tered when the pH is changed. Gaebler (ref . 2) avoids this difficulty by measuring the 
absorbance at 620 mu and at 565 mu, thus compensating for the non-specific absorb- 
ances which are about the same at these two wavelengths. Hamilton (ref. 4) measures 
the absorbance before and after chemical bleaching of the bromsulfaleln by sodium di- 
thlonlte. These last two approaches are important only when the aforementioned inter- 
ferences are present. Another approach which has been used Is to use acetone to pre- 
cipitate the proteins and at the same time extract the dye. The color Is then developed 
In the acetone solution. 

Reagents : Sodium hydroxide 0.1N 
Hydrochloric acid 0. 1 N 

Procedure : 

Collection of specimens: The patient must be fasting (at least 10 hours post-pranT 
dial) and non-icteric, and kept in a supine position in bed for the duration of the test* 
The patient's weight must be known and converted to kilograms (lbs. /2.206). 



112 






2. Draw Into a sterile syringe an exact amount of sulfobromophthaleln so that the 
Injected dose will bo 6 mg. per kilogram of body weight. This volume should be exact 
to the 0. 1 ml, 

3. Then Inject the dye Into an arm vein over a period of 30 seconds. 

4. Start timing the test when half of the dose has been given. 

3. Five minutes lator, from a vein of the opposite arm, withdraw into another 
syringe 3-10 ml. of blood for serum. Take further samples at appropriate times, for 
example at 30, 40 and at 00 mlnutos. For the determination of bromsulfalein clear- 
ance, take samples at about 0, 10, IS and 20 minutes and carefully note the exact 
times. 

Estimation of Bromaulfaleln 

Add 0, 3 ml. of clear, unhemolysed serum to 0.0 ml. of 0, 1 N MaOH In a photo - 
mater. Mix well. Read against a blank prepared in the same way but using 0.0 ml. of 
0. 1 M HOI. Set the blank at T ■ 100 (D . O) using a wavelength of 373 mu. 

Read the retention In per cent from the calibration table which Is based on the ad- 
ministration of 3 mg. per kilogram of body weight. (If the dose was 2 mg. per kilogram, 
multiply by 2. 3 to obtain per cent retention. ) 

Standardization 

Since the results of the test are expressed in terms of per cent retention of the dye, 
It Is obvious that we must determine or assume the "volume of distribution" Into which 
the dye Is diluted. 

The calculation is commonly based on the following assumptions: 

(1) 10% of the body weight Is circulating blood. 

(2) 60% by volume of the blood is plasma. 

■(3) Therefore 6% of the body weight is plasma or 60 ml. per kilogram of body 
weight is plasma. 

Since 6 mg. per kilogram of body weight is injected this is 6 mg. per 60 ml. plasma 
or 10 mg, per 100 ml. of plasma. 10 mg. % Is considered to be the Initial concentration 
of dye in the plasma or 100% retention, 

Bromsulfalein Standard Solution 

Pipette carefully and accurately, 0.1 ml. of 3% bromsulfalein solution Into a 300 ml. 
volumetric flask and dilute to volume with approximately 0. 1 N NaOH. Mix well by in- 
version. 

113 



Calibration Standards 

Into a series of photometer cuvets, transfer 0.0, 0.6, 1,0, 2.0, 3.0, 4.0 and 6.0 
ml. of bromeulfaleln standard solution. Make each tube up to 6,0 ml. with 0.1K NaOH. 
Mix well, then add 0. 6 ml. of normal pooled serum, and mix well again. These tubes 
represent 0, 10, 20, 40, 60, 60 and 100% retention of the dye using the assumptions 
given above. Using the 0.0 tube as a blank T ■ 100 (D = O) record the transmlttance of 
each tube and plot the optical density vs. % retention and tng.% as Illustrated on p. 64a . 
Prepare a calibration graph as outlined on p. 68. 

NOTES 

In this simple analysis the serum must be clear, with no hemolysis, billrublnemta, 
or llpemla. H it Is wished to avoid the effects of such Interferences It is necessary to 
procede with one of the modifications referred to above (ref . 2 or 4) or to use the fol- 
lowing extraction procedure. 

1. Into a 16 ml. centrifuge tube place about 2 ml. acetone. Add 0.6 ml. of serum, 
dropwise with agitation. Mix well. Heat to boiling in a boiling water bath. Cool. Cen- 
trifuge. 

2. Decant the supernatant fluid Into a tube graduated at 7 ml. Add about 1 ml. of 
acetone to the residue in the centrifuge tube, mix well with a wooden applicator stick, 
allow to stand 2 minutes and centrifuge. Decant the supernatant fluid into the 7 ml. 
graduated tube. Repeat once more. 

3. Add distilled water to about 7 ml. Mix well. Then add 2 ml. ether and mix by 
Inversion. Allow to stand 3-5 minutes to separate the phases. Remove the ether 
phase by suction. Repeat once more, 

4. Adjust the acetone water layer to 10. ml. with 0. 1 N NaOH and mix. Read 
against a blank of 3 ml. of acetone plus 3 ml. of water diluted to 10.0 ml. with 
0.1 N NaOH. 



< 



( 



Calculation; 






Multiply the chart reading by 1.64 (10.0/6.5) to obtain % retention or mg.%. 



114 



( 



Interp retation 

Normal Individuals after Injection ol a 5 mg. per kilogram of body weight dose 
have a plasma retention of lea a than 10% at 30 minutes, <7% at 45 minutes and usually 
0-1% at 60 minute a. 

If a 2 mg. per kilogram body weight dose Is used as originally proposed by Rosenthal 
and White (ref. 1) and as readvocated by Lorber and Shay, Gastroenterology 20, 262 
(1952), to minimize entero- hepatic recirculation of the dye,the 30 minute retention will 
be less than 4%. 

The rate of removal of bromsulfaleln by the normal liver is determined by (1) the 
rate of blood flow through the liver, (2) the functional capacity of the polygonal cells of 
the liver, and (3) the degree of patency of the bile duct system. In obstructive Jaundice, 
retention is very high, out of proportion to the actual damage to the functional capacity 
of the liver. Zleve et al. f J. Lab. Clin. Med. 37, 40 (1951) f have devised a correction 
to be applied when the one-minute bilirubin of Watson Is elevated beyond normal limits. 
When corrected in this way, the test more closely estimates the functional capacity of 
the polygonal cells even in obstruction. The diversion of blood from the usual liver 
channels into collateral vessels is an Important cause of high retention not only In cir- 
rhosis but also in viral or chemical hepatitis and also In heart failure. 

It is doubtful that the bromsulfaleln test adds much to the diagnosis or prognosis of 
liver disease In the presence of Jaundice. 



Bromsulfaleln Clearance 

m order to express more clearly the rate of disappearance of the dye, the concept 
of liver clearance (analogous to renal clearance) has been Introduced and applied to not 
only this dye but to other substances such as galactose and hippurlc acid. This approach 
necessarily applies to substances not normally found In serum, since the only way to 
measure the clearance Is by the rate of decrease in serum concentration. Goodman and 
Kings ley (ref. 5) have used this method quite extensively and the description below Is 
based on their work. 

Procedure 

Collection of samples 

1. Injection of dye and the collection of samples Is carried out as indicated under the 
tolerance test with the injection and collection very carefully timed (mid-point time of In- 
jection and collection). The collection of samples should be completed by the end of 20 
minutes. 



115 













































( 
























2.5- 



O 

i- 
z 

Ul 

H2.0 

UJ 



o 



1.0 



•Note: 


Retention at zero time la over 




EPV = 400/14.4 s 2780 ml. 










100% because the plasma vol- 




Cl/V = 2.3 x(2.160 - 1.000)/22.5 = 0. 119/mlnute 




ume is less 


than the assumed 




Cl(ml./mln.) = 0.119 x 2780 


- 331 ml. /mm. 




plasma volume of 4000 ml. 




Cl a (ml./m.in./kg.) = 331/80 = 


4. 14 ml. /mm. /kg. 


■" 




























" utile* 2. 160 


* 144. 0% Hetottw 

= 14. 4 mg. /100 ml. placma 




















- 




































^ 




























^^^^ 


U.I&1BI 
■tlof a : 


1.000 














■X 


U% R 




J. 


J- 




_L _L 


^^^J ' 


1 



10 15 

MINUTES 



20 



25 



Fig. 22. Bromsulfaleln clearance. 



Analysis of sample a 

The samples are analyzed as outlined under the tolerance teat. The results may be 
expressed as In mg. per 100 ml. or In per cent retention. 

Calculation : (Example) (Values In parentheses are obtained by extrapolation) 

Data: Weight of Patient 168.5 lbs. (80 kg.) 
Dose of BSP 400 mg. (80 x 6) 

% 
Retention Log %R 

(1*0 (2.180) 

80 1.902 

43 1.632 

26 1.398 

13 1. 114 

(l.OOO) 

1. Estimated plasma volume = Dosage BSP/Inltial concentration (C,) 

2. Fractional clearance (Cl/V) - 2.3 (log C t - log C|) time (minutes) 

3. Clearance (CI) (ml. /minute) = (CI/V) x V 

4. Clearance (Cl a ) (ml. /minute /kg.) = Cl/kg (body weight) 

In this calculation the initial concentration of the dye (Cj) Is determined by extra- 
polation to zero time and the final concentration Cf is determined by extrapolation to 
logH = 1.0. 

Using the data from the above example: (See Fig. 22, p. 116a) 

1. Estimated Plasma Volume « 400 mg. /14.4 mg.% = 2780 ml. 

(antllog 2.160 = 144.0% retention corresponding to 14.4 mg.% dye concentration) 

2. Fractional Clearance (Cl/V) - 2.3 (2.160 - 1.000)/22.6 minutes 

m 0.119/mlnute 

3. Clearance (CI) (ml. /minute) = 0.119 x 2780 = 331 ml. /minute 

4. Clearance (Cl a ) (ml./mln./kg.) - 331/80 = 4.14 ml. /min. /kg. 



Time after 
injection 


Cone. BSP 





C14.4) 


6 


8.0 


10 


4.3 


16 


2.6 


20 


1.3 


(22.6) 





The following formulae are used: 



The advantages of this procedure are: 



( 



116 






1. The accuracy and precision are both Increased by collection and analysis of a 
series (rather than one) of blood specimens and by the method of calculation. 

2. The method gives an estimate of the plasma volume as well as a numerical 
single value expressing a rate of clearance of the dye. 



3. The test is completed In 20 minutes. 






4. The clearance results do not depend upon the accuracy of measurement of the 
Injected dye (;> 1 though the estimate of plasma volume does). 



Interpretation : 
Clearance 



Normals (28) 
Cirrhosis (18) 
Alcoholics (35) 



Plasma Volume 
Normals 
Cirrhosis 
Alcoholics 









S. 33 i 0. 75 ml. /mm. /kg. 
2.06 >f 0.30 " 
4.45 r 0.63 " 



40.9 $6.16 ml. /kg. 

60.6!? 6.0 

38. 4 2^4. 93 " 



None below 4 ml. /min. /kg. 
None above 3 " 
37% of the values were 

lower than lowest of 

the control group 












117 



CALCIUM 
Serum, Urine, etc. 
References: 






(1) Clark, E. P. and J. B. Collip: J. Biol. Chem. , 63, 481 (1926) 

(2) deLoureiro, J. A. and Q. F. Janz: Blochem. J. , 38, 16 (1944} 

(3) Rehell, B. : Scand. J. Clin. Lab. Invest., 6, 336 (1955) 

(4) Pattern, J. and Reeder, W. : Anal. Chem. , 28, 1026 (1956) 

(6) Nordin, E. C. and R. Fraser: Lancet, 270, 826, 2 June (1966) 

Principle : Calcium Is precipitated as the oxalate, which Is washed free of excess 
oxalate and then titrated In hot acid against KMnO . . 

2 M11O4 / 6 C 2 4 " / 16 fl/ > 2 Mn# / 10 CO a / 8 HjO 

Apparatus : Mlcro-buret 6 ml. in 0,01 ml. divisions. 

Reagents : 

1. Ammonium hydroxide 0. 36 N. Dilute 2 ml. concentrated MH .OH to 100 ml. 
with distilled water. 

2. Ammonium hydroxide 4 N. Dilute 22 ml. concentrated NH .OH to 100 ml. with 
distilled water. 

3. Ammonium oxalate 4% w/t In water. 

4. KMnO. — 0.1 N stock. Dissolve 3. 3 g. pure reagent grade KMnO, crystals 
In 1000 ml. distilled water. Bring to a boll. Cover with a beaker and allow to 
cool and stand in the dark for two to three days or longer before using. 

5. KMnO. — dilute working solution. Dilute 10 ml. of stock 0.1N solution to 
100 ml. with water. Prepare and standardize Just before use. 

6. Sodium oxalate stock 0. 1 |£. Weigh out exactly 6. 7010 g. of pure dry reagent 
grade sail, dissolve and dilute to 1 liter with IN B^SO^ This solution Is stable 
for several months. 

7. Sodium oxalate working standard. Dilute 10.00 ml. 0.1 N stock solution to 
100 ml. with 1 N H SO . Mix well. Prepare weekly. 



118 



( 









8. Sulfuric acid 1 N. Dilute 3 ml. concentrated H 2 S0 4 to 100 ml. with distilled 
water. Mix well. 

9. Phenol red - approximately 0.04% in water. 

10. Acetic acid 10%. Dilute 10 ml. of glacial acetic acid to 100 ml. with distilled 
water. 

PRECIPITATION 

Serum: 

1. Clean a 12 ml. heavy walled conical centrifuge tube by filling with dlchromate 
cleaning solution and allowed to stand for at least 10 minutes, preferably over- 
night. Rinse thoroughly with distilled water and drain. 

2. Add 2. 00 ml. cell-free serum to the tube using an Ostwald plpet. 

3. Add 2 ml. distilled water. 

4. Add 1.0 ml. ammonium oxalate 4% solution. 

5. Mix well by tapping and allow to stand for one hour . 

6. Centrifuge at highest speed for 10 minutes (do not use an Inclined head), invert 
quickly and drain for 6 minutes, inverted on a piece of clean filter paper; then 
dry the mouth of the tube with a piece of filter paper. 

T. Blow Into the tube about 1. 6 ml. of 0. 36 N NH^OH breaking up the precipitate. 
Rinse the sides of the tube with 1. 5 ml. more of the solution. 

8. Re centrifuge, invert quickly and drain 5 minutes, drying the mouth of the tube 
after draining, as before. 

9. Repeat washing, centrtfugatlon and draining. 
Urine : 

1. Plpet exactly 1-5 ml. (depending on tiie expected calcium content) of urine into 
a conical tube. 

2. Add 1 drop of phenol red Indicator and then 4 N NH^OH drop by drop until the 
indicator Is pink. 

3. Immediately add 10% acetic acid drop by drop until the indicator Just turns 
yellow. 

119 



4. Add 1 ml. of ammonium oxalate 4,%. 

6. Mix well and let stand 1-2 hours at room temperature. 

6. Proceed with centrifuglng and washing as In serum calcium but wash the preci- 
pitate two extra times. 

Food, Tissue and Feces: 

1. Ash an appropriate dried aliquot of sample at 600°C. , for several hours, to a 
white ash. ff a dark ash Is obtained, add a few drops of concentrated HC1, dry 
and relgnlte at 600°C. 

2. Dissolve the ash In a drop or two of concentrated HCl. 

3. Transfer quantitatively and bring to volume In a SO ml. volumetric flask. 

4. Proceed with precipitation and washing as In the urine calcium procedure, 
using a 5. 00 ml. aliquot. 



TITRATION OF STANDARD 

Into a 12 ml. centrifuge tube pipet exactly 2. 00 ml. 0.01 N sodium oxalate solu- 
tion, using an Ostwald pipet. Heat for two minutes in a boiling water bath and titrate 
while still hot (keep in a beaker of hot water) with Q. 01 N (approximately) KMn0 4 solu- 
tion to the first permanent pink; titrate slowly at first, allowing each drop to react 
before adding the next. 



Correction factor = 2 ml. /ml. KMnO^ used 

or should be between . 05 and 1 . 
tlon by addition of more water or more KMn0 4 



The factor should be between 0.05 and 1. 05. If It Is not, adjust the KMnO. solu- 



TITRATION OF UNKNOWNS 

1. Add 2.0 ml. 1 N H 2 S0 4 , tap gently to break up the precipitate. 

2. Heat for 2 minutes In the boiling water bath, until all of the precipitate ts 
dissolved. 

3. Titrate, using the precautions noted above, to the first permanent pink. 

120 



( 









CALCULATIONS 

One (1) ml. of 0. 01 N sodium oxalate solution {or of 0, 01 N KMnOA 1b equivalent 
to 0.2 mg. of calcium. 

1.00 ml. x 0.01 N a 0.01 m£q. 

0.01 mBq. x 40 (at. wt. Ca) = Q.amg. Ca. 

2 (valence of Ca) 

0.2 x ml. permanganate (0.01 N) x 100/2 a mg. Caper 100 ml. serum 
or 10 x (ml. permanganate) x (correction factor) s mg% Ca. 

Example: 2. 00 ml. 0.01 N sodium oxalate required 1.080 ml. of approximately 
0. 1 N KMn0 4 for titration. 

Factor = 2.00/1.98 > 1.01 

The calcium oxalate precipitated from 2.00 ml. of serum, titrated as above, re- 
quired 1.000 ml. of mis permanganate tor titration. 

10 x 1.000 xl.01 s 10.7 mg. Caper 100 ml, serum 

Urine and Other Samples : 

The calculations are very similar, except that the degree of dilution and size of 
the sample most be considered. 

Motes; 

1. Extreme care Is required in mixing, washing and draining, to avoid loss of 
some of the precipitated calcium oxalate. 

2. The solution, during the titration, most be kept between TO and 76°C. to insure 
a stoichiometric reaction. A brown color, (MnO„) obtained by titration at tower tem- 
peratures, or by too rapid initial titration, will result in inaccurately high values. 

3. When the firs^drop of KMnO^ is added the pink color persists for some time. As 
soon as catalytic amounts of manganous (Mn^r) gaits are formed (see reaction p, 118) 
the reaction proceeds more rapidly. 

4. In the analysis of urine it is advisable to use fresh samples if at all possible. 
For 24-hour collections and for brief preservation add 4 ml. glacial acetic acid per 
200) mL of urine. Measure the total volume before allquottlng. The urine calcium, de- 
fe mritmfr i iw ti [jfl; less; specific dee to wigfeuie au tWtftmc flap being adaotrbed! to; the: precipitated 
caJcitam imnihhn ({Hurfkiaen 






6. Other methods which might be used Include: 

(a) Oxalate precipitation methods using permanganate with added MnSO^ to 
catalyze the reaction at room temperature (Ref . 2) with subsequent analysis 
by titration lodlmetiy or photometry. 

(b) Chelatometrlc methods based on the formation of an extremely stable soluble 
calcium complex with ethylene -diamine -tetraacetlc acid (EDTA) together 
with the use of specific Indicators (Ref. 3 and 4). 



INTERPRETATION 

Serum Calcium: 

All of the calcium in the blood is confined to the plasma. The normal range for 
serum total calcium Is between 9 and 11 mg. per 100 ml. serum. (5 mEq. per liter; 
2. S mil. per liter), m Infants (especially premature) with adequate Vitamin D Intake 
the level may be as high as 12 to 13 mg. %. 

About 1/3 of the calcium of the serum is bound to protein and is "Inactive" physio- 
logically. The remainder is composed of ionized (mostly) and unionized (citrate T) cal- 
cium, only the ionized portion is involved In the control of serum calcium level and in 
muscle irritability. The level of total serum calcium is affected by: 

(1) Deficient Intestinal calcium absorption. 

(2) Alteration in parathyroid hormone secretion. 

(3) Changes in serum inorganic phosphate. 

(4) Alteration in plasma proteins. 

A recent formula (Ref. 5) for the proportion bound to protein is: 

Protein-bound Calcium - (7.6 x albumin) / (2.9 z globulin) - 3.1 
in which protein-bound calcium is expressed In per cent of total calcium and a l bu m in 
and globulin are expressed in g. per 100 ml. serum. For example: 

A serum gave the following values — Total calcium 10.8 mg.% 

Albumin 4.6 g.% 

Globulin . 2. 1% 

Protein BdL Ca = (7.6x4.6) / (2.9x2.1) - 3.1 
= 35/6.1-3.1 
= 38% 
10.8 x 0.38 = 4. 1 mg.% calcium is protein-bound 
6.7 mg.% calcium is diffusible. 



122 






Decreases In Scrum Calcium arc neon in: With Values given as: 

Hypoparathyroidism Lees than 6 mg.% 

Rickets (Vitamin D deficiency) (children) Usually 8-9.6 mg.% 

Steatorrhea (as In sprue, coellac disease) Usually 8 - normal 

Renal failure (secondary to elevated PO.") As low as 6 mg.% 

Tetany may be seen In patients with decreased total serum calcium with abnormal 
reflexes below 8 mg.% and carpopedal spasm below 7 mg.%. These values are modified 
to some extent by variation in serum protein level and actd-base balance In so far as 
these modify the proportion of the total calcium present as ionized Ca^, since it is 
the level of Ionized calcium which influences the Irritability of muscle. The overall 
irritability of muscle Is also Influenced by many other factors as well. 

A decrease In serum calcium Is never the cause of excessive bleeding. The serum 
calcium level required for coagulation is so small that It is always present In excess, 
m massive transfusions, citrate may bind enough Ca^r to affect coagulability if com- 
bined with other effects. Oxalate and citrate both may cause tetany by binding Ca^. 

Increases in Serum Calcium are seen In : Representative Values : 

Hyperparathyroidism 12 - 20 mg. % 

Hypervitaminosls D 12-17 mg. % 

Multiple myeloma 12 - 17 mg. % 

In most other diseases, Including bone disease, the serum calcium Is maintained 
within normal limits due to very close physiological control and the large reserve 
storehouse of readily available calcium — the bones. 

Calcium Excretion : 

On the ordinary diet containing 0.7 - 0. 9 g. of calcium dally, about 75% of the cal- 
cium appears In the feces, the remainder appearing In the urine. The proportion de- 
pends somewhat on calcium binding substances present in the diet. 

There is an increased urinary excretion of calcium in hyperparathyroidism and In 
multiple myeloma. A decreased urinary excretion of calcium is seen In rickets due to 
Impaired Intestinal absorption of calcium. 

Calcium Balance Tests: 

In some disease states the net loss of calcium from the body may be bo slow (over a 
period of 20 to 30 years In the case of osteoporosis) that the serum calcium values or 
the urinary excretion rate may not seem to be affected even though the excretion Is 
greater than the intake. In these cases a Calcium Balance may be carried out as follows: 



123 



The patient Is placed on a low calcium diet containing about 100 - 150 mg. of cal- 
cium and 500 - 700 mg. of phosphorus dally, and three days arc allowed for the patient 
to become adjusted to the diet. A feces marker (carbon, carmine, or gentian violet) 
Is given before breakfast the day collections arc started. Three to four day collections 
are made at the end of which another marker la given before breakfast at which time 
urine collection ceases for that period. Feces collection Is made from marker to 
marker, Including In the test the specimen containing the first marker but excluding 
that containing the second marker. 

The mine and fecal calcium Is determined and the average dally excretion com- 
pared to the known Intake. 

Caldnm Infusion Tests: (Ref . 5) 

In order to avoid the long tedious procedures Involved In calcium balance studies, 
the rate of excretion of calcium In the urine after infusion of 400 - 1200 mg. of calcium 
as the gluconate (or other non-lnitant salt of calcium) has been studied and has served 
as an index of therapeutic effectiveness In osteoporosis, hypoparathyroidism and sev- 
eral other diseases. (Perloff, W.H. , J.H. Boutwell, Jr., and R.H. Haas: Jf. Aroer. 
Ger. Society, 4, 760 (1966). (Shilling, A. , and Lazlo, D. , Rate of urinary calcium 
excretion following Intravenous infusion. Proc. Soc. Exper, Biol. 4 Med. , 78, 286 
1961.) 















( 









CALCIUM BALANCE DIET (MODIFIED AUB DIET) 

Place patient on a diet containing 100 mg. of calcium per day for 6 days, collect 
24-hour samples of urine for the last 3 days of the diet period. Pool and analyze for 
calcium. 

An excretion of 300 mg. or less of calcium for the 3-day period is considered 
normal. In hyperparathyroidism, considerably over 300 mg. will be excreted. 



SAMPLE DIET 



Allow 



Soup 

Meat, Fish, Poultry 



None 

2 of following dally 

60 grams lean beef 



50 


n 


chicken 


60 


» 


lamb 


60 


■1 


mackeral 


65 


it 


lean veal 


80 


it 


turkey 


70 


ii 


halibut 


60 


it 


codfish 



Avoid 

Omit entirely 
Use no other 
meat, fish 
or poultry 






Cereal, Eggs and Milk 



Omit entirely 



Vegetables 


So 


r folic 


wing dally Use no other 


0.009 gram calcium 


40] 


grams peas vegetables 




45 


ii 


asparagus 




65 


ii 


summer squash 




60 


it 
it 


winter squash 
potatoes 




75 




80 


ii 


fresh tomatoes 




00 


ii 


cucumbers 




100 


ii 


corn 




100 


ii 


egg plant 




130 


ii 


fresh tomato Juice 


Potato Substitute 


1 of following dally Avoid all other 


0.003 grams calcium 


25 


grams 


dry rice 




15 


it 


spaghetti 




15 


>t 


macaroni 




30 


it 


hominy 



125 



CALCIUM BALANCE DIET (MODIFIED AUB DUST) (Coot.) 

Allow Avoid 



< 






Fruits 


4 of following dally 


Avoid all other 


0.007 grams calcium 


50 grams canlalopc 






50 " cherries 






50 " grapefruit 
50 " plums 
50 " pineapple 
60 " apricots 






60 " pears 

80 " grapefruit Juice 






90 " peaches 






100 " bananas 






100 " apples 






100 " watermelon 






100 " fresh tomato Juice 




Beverages 


Fluid Intake should be 
constant throughout 
test period. 


Avoid all other 




1 cup coffee at breakfast 






1 cup tea at noon & evening meal 




Water may be bad between meals 


Bread 


30 grams bread each meal 


Avoid all other 


0. 008 grams calcium 




foods 


Miscellaneous 


Sugar as desired 





< 









12t 






CALCIUM 
Serum 

Reference: 

,T crro, P.V. , and Ham, A.B. , Am. J. Clin. Path. , 28, 208 (PJ57). 

Principle : 

Calcium Is precipitated as the Insoluble salt of chloranlUc acid, ft Is washed free 
of excess chloranllic acid, using lsopropyl alcohol. The precipitate of calcium chlor- 
anflate is then dissolved in Na EDTA (ethylenedlaraine tetra-acetic acid, tetra sodium 
salt). The resulting pink solution is compared photometrically against similarly pre- 
pared standards and blanks. 



Reagents : 

1. Chloranllic acid (1%). Dissolve 1 g. of chloranllic acid m 60 ml. of distilled 
water containing 7 ml. of IN NaOH. Mix and dilute to 100 ml. with distilled water. 

2. Na 4 EDTA (5%) (ethylenedlamtnetetraacetlc acid, tetra-sodlum salt). Dissolve 
five grams of Na^EDTA In 100 ml. distilled water. 

3. lsopropyl alcohol 50%. Mix equal volumes of lsopropyl alcohol and distilled 
water. 

4. Calcium standard. Weigh out exactly 0.2497 g. reagent grade calcium carbo- 
nate (CaCO ). Wash Into a 1 liter volumetric flask. Add gradually, 10ml. of 1 N HC1, 
using it to rinse down the Bides of the flask. When solution la complete, dilute to vol- 
ume and mix. 1 ml. = 0. 1 mg. calcium. 

Procedure : 

Into four 12 ml. heavy duty, conical, pyrex centrifuge tubes cleaned as outlined 
on o. 118, add reagents and proceed as outlined. 

Blank Std 1 Std^ Unkn. 



H z O 

Standard Calcium 

Serum 

Chloranllic acid 


(Ml. to be added) 
2 1 
1.00 

1 1 






2.00 

2.00 

1 1 

2. Mix the contents of each tube wed and allow to stand at room temperature for 
at least 30 minutes. 

3. Centrifuge, decant and drain on a filter paper for 5 minutes. Wipe the Up of 
the tube free of the last drop. 

4. Blow In 4 ml. of 50% lsopropyl alcohol so as to break up the precipitate. Mix. 

5. Centrifuge, decant, and drain as in step 3. 

6. Add 1 drop of distilled water to each tube. After standing one minute gently tap 
the tube to break up the precipitate. 

127 



T. Add 4.00 ml. of Na.EDTA to each tribe, mix well and after complete clearing 



(about S minutes) compare photometrically at 620 m agabut the blank eet at 100% T. 
Calculations: 



Ca (mg.%) = (P m /D^ a C, a (100/*) 
(ForStdj) - 0V*V a t.l a (lOO/* 






(Forftd^ ■ OW * M 









Interpretation ; 

Seeprerlooa method. 






















































( 
























128 



CAUBON DIOXIDE OF PLASMA CAPACn Y AND CONTENT 



Reference : 

VanSlyke, D. D. , andCullen, O. E. , J. Biol. Chem. , 30, 289(1917) 

Principle : 

The carbon dioxide capacity In determined after, equilibrating plasma In a 300 ml. 
separatory funnel filled with ana: mixture whoee carbon dioxide tension approximates 
that of normal arterial blood. By this technique the sample of plasma combines with 
as much CO» as It le able to hold under normal CO, tension in the circulating plasma 
(40-45 mm. Hg.). 

The carbon dioxide content determination requires special care In plasma collec- 
tion and preservation to Insure that the content of gases does not change until the anal- 
ysis Is completed. In this determination we attempt at all times to keep the blood at the 
CO„ tension that existed In the body. The blood should be collected without exposure to 
air and transferred immediately to a tube under mineral oil. For details read further 
under methods of collection and preservation of specimens. If blood is collected In a 
"Vacutalner" tube, mineral oil is not necessary and this technique requires centrlfuglng 
without removing the stopper, then rapid removal and measurement of the sample. The 
tube Is then restoppered Immediately, thus preserving the sample for a second deter- 
mination or a pH determination if It is also desired. 

ACID-BASE BALANCE - DISCUSSION 



Reference : 

Levinson and MacFate: pp. 176-181 
Peters and Van Slyke: Vol. I, Chapter IS 

There are three buffer pairs In the blood: (1) the primary -secondary phosphates/ 
(Na 2 HPO./NaH P0 4 ) which have little clinical significance In maintaining a const, nt 
pH in blood; (2) acidic -basic proteins, particularly hemoglobin, which act primarily 
to prevent carbon dioxide from changing the pH of venous plasma more than about 0.03 
pH units from that of arterial plasma, and to aid In the procesB of transportation and 
liberation of carbon dioxide; and (3) < t rbonic acid -bicarbonate (NaHC0 3 /H 2 C0 3 ) which 
is the chief buffer against strong acids or bases. 

The hydrogen ion concentration of the blood Is directly related to the ratio of bi- 
carbonate to dissolved carbon dioxide concentration, by the equation: 

pH = pR^ / log (NaHCOa) Thla lB o^ "buffer equation" of Henderson 
(H 2 C0 3 ) and Hasselbalch. 

Therefore the hydrogen Ion concentration (acidity) of the blood increases if the 
denominator of this fraction is increased, or if the numerator Is decreased. The acidity 

129 



decreases If (he denominator decreases or if (he numerator Increases. The body mech- 
anisms (lungs, kidneys) attempt always to maintain a constant hydrogen Ion concentra- 
tion by making the numerator and denominator vary together, thus keeping a refctlvety 
constant quotient or ratio. When a strong acid, such as B-hydroxybuty*ic acM o# acetfrp 
acetic acid, is introduced Into the- blood,, it reacta with the bicarbonate present to 
form the sodium salt of the stronger acid, plus carbon dioxide. Then enough of the 
latter is excreted by the lungs to restore the above ratio nearly to normal. Thus the pH 
of the plasma is maintained relatively constant, though the concentrations of COg and 
bicarbonate in It are decreased. 

Conditions which upset the ratio of H 2 C0 3 to HCO3 can be grouped under four heads: 

1. Primary alkali (bicarbonate) excess . This condition Is known as "alkalosis. " 

It may be caused by the Ingestion of large amounts of bicarbonates or other alkalies, or 
by the loss of HC1 from the stomach in extensive vomiting, as In pyloric obstruction or 
in toxemias of pregnancy. This acid Is formed by the stomach from NaCl, leaving 
NaHC0 3 In the blood. If then the HC1 is not reabsorbed, but Is vomited, there will 
come to be an excess of bicarbonate In the blood. 

2. Primary alkali deficit (known as "acidosis"). B Is found particularly In diabetes 
mellltus and in terminal nephritis; also In severe diarrheas of Infants, In starva- 
tion, and In ether or chloroform anesthesia. S Is due either to loss of NaHCOg, or to 
formation of sodium salts of non-volatile acids, which are not available for buffer action. 
These are excreted by the kidneys, which attempt to conserve some of the sodium by 
formation of ammonia from glutamlne (and other amino acids) to replace sodium. Ex- 
cretion of salts Is accompanied by loss of fluids and dehydration. Therapy : Administer 
physiological saline, glucose and insulin (in diabetes) to prevent formation of acids. 
Bicarbonate administration Is usually not necessary and may be harmful If carried too 
far. 

3. Primary CO excess. Rare; occurs in morphine poisoning and In Ayerza's 
disease - fibrosis of the lungs . 

4. Primary CO a deficit . Caused by over-ventilation of lungs. Rarely this is of 
clinical significance. It occurs in O z deficit, fevers, encephalitis with affection of 
respiratory center; and In hysteria. 

The bicarbonate content of blood plasma Is known as its "alkali reserve. " It is 
easier to determine total CO z obtainable by mixing plasma with acid; so alkali reserve 
Is expressed as the number of ml. of COg (at 0° C. and 760 mm. Hg.) that can be 
obtained from 100 ml. of acidified plasma. 55 to 75 ml. per 100 ml. of plasma or 
55 to 75 volumes per cent is normal. Normal pH of plasma is 7. 3 to 7. 6. The pH 
seldom goes below 7.3 (acidosis remains "compensated") until the alkali reserve 
gets down below 40 volumes per cent. Hypernea appears when the value falls to half 
of normal (moderately Bcvere acidosis). Below 30 volumes per cent, acidosis is 
Severn, though recovery may occur with values as low as 10 to 15 per cent. Such 
cases however, usually terminate in death. 

A. VOLUMETRIC PROCEDURE 

Pri ncip le: 

Of the CO that may be obtained from blood scrum the greater part is present in 

130 












the form or the bicarbonate Ion (IICOjj). Before Oils poteiu.*i* carbon dioxide can be re- 
leased for measurement, It must be freed from combination. Lactic acid, a stronger 
add than carbonic. Is used to convert the bicarbonate Ion to carbonic acid and the gas 
(COJ Is extracted under vacuum. Complete removal is not possible with one extrac- 
tion out If the amount of solution and the ratio of liquid phase to gas phase is kept con- 
stant, the residue of CO, remaining in solution can be calculated from the solubility 
coefficient and the temperature and pressure. At the same time correction is made 
for the dissolved oxygen and nitrogen that are released by the extraction. The actual 
volume of COg extracted can be obtained by absorbing the C0 2 with alkali such as 
NaOH. 

Apparatus : 

Van Slyke Cuilen Volumetric Carbon Dioxide Apparatus. See Fig. 23. p. 132a. 

Reagents : 

1. Lactic acid 0.1N (approx.). Dissolve 1 ml. of lactic acid sp. gr. 1.20 in 
enough water to make 100 ml. of solution. Add enough phenol red indicator solution to 
color. 

2. 1 NNaOH. To 5.5 ml. of 18 N NaOH add with mixing enough distilled water to 
make 100 ml. of solution. 

3. Caprylic alcohol (Methyl n-hexyl carbinol) In dropping bottle. 

4. Mercury In dropping bottle. 

Procedure : 

Note: You should become acquainted with the position of stopcocks and leveling 
bulb and also the feel of the stopcocks In order to control correctly the flow of mercury 
and solution In the apparatus before starting the determination. 

1. The entire apparatus, Including the capillaries above the upper stopcock E, is 
filled with mercury. 

2. Add one drop of caprylic alcohol to cup; open the lower stopcock, and by control- 
ling the upper stopcock, allow the caprylic alcohol to run down Into the capillary above 
the upper stopcock. 

3. Add a few drops of mercury over the caprylic alcohol In the capillary, and seal 
stopcock with mercury, allowing the alcohol to go into the pipet. 

4. Two ml. of lactic acid is placed in the cup B (an approximate measurement; use 
marking on the cup). 

5. One ml. of serum (measured with a pipet calibrated between marks) is run into 
the cup under the lactic acid without mixing, or rubuer-Upped pipet Is used. 

6. With the leveling bulb in low position (2) , open stopcock £ and control the deliv- 
ery of serum and lactic acid by means of stopcock F. The lactic acid Is run In until the 
mercury reaches the 2. 5 ml. mark on pipet. Close both stopcocks and remove excess 
lactic acid from cup. Seal with mercury. 

7. With the leveling bulb lowered to very low position 3, open the lower stopcock 
and draw the level of the mercury (not the aqueous mixture) to the 50 ml. mark which 
is Just above the lower stopcock. 



131 






8. Place leveling bulb securely In position 2 and remove apparatus from stand. 
Shake, without inverting, for two minutes to liberate the gnu. Place the apparatus back 
In stand carefully and allow to drain for 30 seconds. 

9. Draw aqueous layer (but no gas) as completely as possible Into chamber D by 
opening lower stopcock and lowering leveling bulb. It is better to control the flow of 
fluid by means of the lower stopcock. 

10. Close lower stopcock and place leveling bulb In position 2. 

11. Open the lower stopcock to the side arm C and allow mercury to rise slowly In 
the apparatus. Raise leveling bulb and place at such a height that the surface of the mer- 
cury Is at exactly the same level as the mercury In the apparatus. The small amount of 
aqueous fluid can usually be neglected. If more than 2 or 3 cm. of solution Is above the 
mercury, the height of the mercury In the leveling bulb should be raised l/13th of the 
height of the aqueous column above the inside mercury level. Close lower stopcock and 
place the leveling bulb back In position 2. Read the volume of gas (at the aqueous men- 
iscus - not at the mercury level); call the volume V; record the temperature of the en- 
vironment and barometric pressure at the time of determination. 



When CO ? In plasma or serum Is determined, the reading of the total gas volume 
may be taken as the finish of the determination. A certain amount of air carried Into 
the apparatus dissolved in the plasma and in the 0. 1 N lactic acid is mixed with the C0 3 , 
but the correction necessary for this may be calculated from the solubility of air in 
water at room temperature, and In plasma at 38° C. , with sufficient accuracy for 
most purposes. The air correction bo calculated is given in the second column of the 
table. 

Factors for calculating CO. content determined by volumetric apparatus with 
blood or plasma samples of 1 ml. (from Van Slyke and Stadle) are shown In this table: 



( 



Temperature 


Air In extracted gases from plasma 


Factor by which ml. of 




and water. 


Subtract from observed 


CO« extracted from 1 ml. 
of plasma of blood Is 


o c 


sir / C0 2 


volume If C0 9 and air 




are measured together. " 


multiplied to give Vol- 








umes of co 2 




22 




0.04S 


98.0x1 


B*/760 


23 




0.04S 


96.4 x 


ti 


24 




0.04S 


94.8 x 


ft 


25 




0.044 


94.2 x 


ti 


26 




0.044 


93.6 x 


ii 


27 




0.044 


93. lx 


ii 


28 




0.043 


92. 4 x 


ii 


29 




0.043 


91.8 x 


ii 


30 




0.043 


91.2 x 


ti 


31 




0.043 


90.6 x 


it 


32 




0.042 


90.0 x 


ii 


33 




0.042 


89. 4 x 


t> 


34 




0.042 


88.8 x 


ti 



*B = observed barometric pressure 



132 








>Vbfcjme of COg gas 






^Column of woter 
\eveHnQ of mercury 

*i tevelng hu* 



PosHion3 should 
be 80 cm. below 
Position 2. 




rifttnt 13. V«a Sljrke-Cullen Volumetric C0| App*ir*iu« ( 



111ft 




































( 



Chart for Rapid Calculation of Carbon Dioxide Valu es 








B.P. = 


753-767 mm. 


Hg. ; Temperature 


- 23-26°C. 




Volume of gas 




meq CO z 


Volume of gas 




meq C0 2 
per liter 


as read 


Vol. % 


per liter 


as read 


Vol. % 


1.00 


90.3 


39.3 


.60 


52.3 


22.7 


.99 


89.3 


38.8 


.59 


51.3 


22.3 


.98 


88.4 


38.4 


.58 


50.4 


21.9 


.97 


87.4 


38.0 


.57 


49.4 


21.6 


.96 


86.5 


37.6 


.56 


48.6 


21.1 


.95 


85.5 


37.2 


.55 


47.5 


20.7 


.94 


84.6 


36.8 


.54 


46.6 


20.3 


.93 


83.6 


36.3 


.63 


45.6 


19.8 


.92 


82.7 


36.0 


.52 


44.7 


19.4 


,91 


81.7 


36.6 


.51 


43.7 


19.0 


.90 


80.8 


35.1 


.50 


42.8 


18.6 


.89 


79.8 


34.7 


.49 


41.8 


18.2 


.88 


78.9 


14.3 


.46 


40.9 


17.8 


.87 


77.9 


.13.9 


.47 


39.8 


17. S 


.86 


77.0 


33.5 


.46 


39.0 


17.0 


.85 


76.0 


33.0 


.46 


36.0 


16.6 


.84 


75.1 


32.7 


.44 


37.1 


16.1 


.83 


74.1 


32.2 


.43 


36.1 


15.7 


.82 


73.2 


31.8 


.42 


35.2 


16.3 


.81 


72.2 


31.4 


.41 


34.2 


14.9 


.80 


71.3 


31.0 


.40 


33.3 


14.4 


.79 


70.3 


30.6 


.39 


32.2 


14.6 


.78 


69.4 


30.2 


.38 


31.4 


19.7 


.77 


68.4 


29.7 


.37 


30.6 


13.8 


.76 


67.5 


29.3 


.36 


29.6 


12.8 


.75 


66.6, 


28.9 


.35 


28.6 


12.4 


.74 


65.6 


26-5 


.34 


27.6 


12.0 


.7» 


64.6 


29.1 


.33 


26.6 


11.7 


.12 


63.7 


af.7 


.32 


25.7 


11.2 


.71 


62.7 


27.3 


.31 


24.7 


10.7 


.70 


61.8 


26. V 


.30 


23.8 


10.3 


.69 


60.8 


26. 4 


.29 


22.8 


9.9 


.60 


59.9 


26.0 


.28 


21.9 


9.6 


.67 


68.9 


25.6 


.27 


20.9 


9.1 


.66 


58.0 


25.2 


.26 


20.0 


8.7 


-65 


57.0 


24.8 


.25 


19.0 


8.3 


-64 


56.1 


24.4 


.24 


18.1 


7.9 


.63 


55.1 


24.0 


.23 


17.1 


7.4 


.62 


54.2 


23.6 


.22 


16.2 


7.0 


.61 


63.2 


23.1 


.21 


15.2 


6.6 



133 



Volume of 


gas 






mcq COg 


as read 




Vol. % 




per 


liter 


.20 




14.2 






6.2 


.19 




13.3 






5.8 


.18 




12.4 






5.4 


.17 




11.4 






5.0 


.16 




10.5 






4.0 


.15 




9.5 






4.1 


.14 




8.6 






3.7 


.13 




7.6 






3.3 


.12 




6.7 






2.9 


Assumption 


8: 








1. 


Barometric 


pre 


aaure 


varle 


2. 


Tern 


peratur 


e varies 1 


)etwee 












itween 753-767 mm. Hg. 
3° and 25° C. 

Note : 

If plasma Is allowed to stand exposed to the air while In contact with its erythrocytes, 
even more drastic changes will occur due to the Hamburger effect (the chloride shift). 
(Such changes are Irreversible If the red blood cells are then removed.) This may best be 
explained by the following logical (not chronological) series of events. 

La tissues: 

1. CO, having been formed by the metabolic activity of the cells, passes through the 
intra-cellular fluid and then into the plasma. From the plasma the CO« passes into the 
red blood cell. 

2. In the red blood cell, It forms HgCO, by reacting with H„0, catalyzed by carbonic 
anhydrase. H 2 / CO z * H 2 C0 3 

3. The newly formed H 2 C0 3 produces Uttle or no change In the erythrocyte pH be- 
cause of the availability of potassium Ion (KO liberated by the conversion (by loss of Oj>) 
of the stronger acid HHbOg (oxyhemoglobin) to the weaker acid HHb (reduced hemoglobin). 

4. The newly formed KHCOg ionizes to form K^ and HC03~. Most of the HCO3" dlf-. 
fuses back into the plasma. 

5. Since yJ' as a cation is unable to diffuse with HC0 3 ", an equivalent quantity of CI" 
diffuses from the plasma into the erythrocyte to maintain electrical neutrality. 

In the lungs : 

The reverse of the above events takes place. 

1. HKb accepts oxygen and becomes a stronger acid, HHb0 2 which recaptures K^ and 
reforms HgCO, which breaks down to form C0 2 and water. 

2. The C0 2 diffuses from the cell into the plasma and out Into the alveolar spaces, 

3. Then more HC0 3 " diffuses back into the cell and CI" diffuses out, 

4. Thuu the overall effect is the loss of CO„ to the lungs from the plasma HCOj 
via the red cells (where HC0 3 ~ becomes COo). 

134 



( 



When C0„ In plasma Is determined without the introduction of alkali, It la not nec- 
essary to wash the apparatus out between analyses. The slight amount of CO- remain- 
ing dissolved in the form of acidified solution widen adheres to the chamber walls Is 
negligible. After the measurement the contents are expelled into waste Jar by elevat- 
ing the leveling bulb and turning stopcock as to connect with waste jar. Air diffuses 
through rubber tubing slowly and may gradually accumulate In the apparatus. A blank 
extraction of 2. 5 ml. of the lactic acid should yield no more extracted gas than the vol- 
ume indicated in the second column of the above table. 

12. The best procedure Is to absorb the CO, by alkali. This absorption must be 
done when whole blood is analyzed. After the measurement of total gas volume, V| , 
the leveling bulb is lowered so that a partial vacuum Is obtained, the gas space being 
increased to about 5 ml. in the chamber. The lower stopcock is now closed and 2 ml. 
of 1 N NaOH solution (see "CommentsJ' below) Is placed in the cup of the apparatus. 
One ml. of alkali Is allowed to flow slowly into the chamber, at least 30 seconds being 
taken for its admission. After the alkali is admitted, the upper cock Is sealed with 
mercury. The solution is now allowed to drain for one minute. (Do not shake.) 

13. The gas volume is now brought to atmospheric pressure in the same manner 
as before and its volume is read. Call this volume V„. The difference, Vj-V,, Is the 
ml. of carbon dioxide obtained from 1 ml. of serum at t°C. and B mm. Hg. pressure. 
This value (ml. of CO„) is multiplied directly by the factor Indicated in the third col- 
umn of the above table. 



Calculation: 
Example: 


If Vj 

V 2 
V l- V 2 


= 0.72 ml. 
m O.OS ml. 
= 0.67 ml. 




Volumes % CO 


Preparation of 


apparatus ; 





Barometer 

Factor 

t°C 



750 mm. Hg 
= 94,2 x 750/760 
= 25° C. 



= 0.67 x 94.2 X 750/760 
= 62. 3 Volumes % 



If this is kept clean, with well greased stopcocks, the only preparation necessary 
is to test for leaks. This test should be routine, and should never be omitted. It is per- 
formed by sealing both capillaries of the top stopcock with mercury, closing it, and 
drawing a vacuum to the 50 ml. mark. The Btopcock grease is not strong enough to 
prevent leakage unless the capillaries and the bores in the plug are filled with mercury. 
Therefore, it la important that the stopcock be sealed with mercury before it is put 
under reduced pressure. After being drawn to the 50 ml. mark, the mercury is then 
allowed to rise to the top cock and to strike it gently. A sharp click should be produced. 
A muffled or soft click is an indication of the presence of air. 

Precautions to be observed in handling blood gas apparatus : 

1, When the apparatus is not in use the reaction chamber should be filled with water. 

2. The stopcocks should be well greased (avoid excess) so that the flow through the 



135 



system can bo controlled smoothly. Do not allow the stopcocks to be In contact with 
alknline solutions longer than la necessary. The Instructor will demonstrate the proper 
method of greasing the stopcocks. The stopcock plug must always be handled a short 
distance above a desk (preferably wooden) so that there Is no chance of Its slipping from 
greasy fingers onto the floor. If the plug falls a few Inches onto the desk it will not 
break, 

3. Mercury is heavy. When you handle the leveling bulb make sure that y<> : have a 
good hold on it. When you set the leveling bulb down In the Iron ring holders do it with 
care and make sure that It Is secure before you let go. 

4. Mercury is expensive. Please take care not to waste it and particularly avoid 
spilling of mercury into the sinks. If any mercury is spilled on desk, floor or sink, 
recover it and place it in the waste Jar provided with each apparatus. This mercury is 
not thrown out. It is carefully cleaned and used again, 

5. Mercury forms amalgams with the noble metals. Therefore It Is suggested that 
all Jewelry, Including rings, wrist watches, and gold-trimmed fountain pens be removed 
from your person while working with this apparatus. 

6. Please leave the apparatus at the end of the day filled with water as you found It 
at the beginning of the period. 



< 



Interpretation: 

See Appendix for discussion of electrolyte balance. 

Note : For rapid calculation of 0O 2 in volumes % and meq. /liter when the measurements 
are made at close to 760 mm. Hg. and 24°C. see table p. 133. 






























136 






CARBON DIOXIDE 

B. MANOMETRIC PROCEDURE 

Principle : 

Carbon dioxide 1b liberated from plasma by lactic acid (along with small amounts 
of other gases, such as 2 and N„). The pressure at a constant volume (2 ml.) Is 
measured before and after absorption of the CO„ with NaOH. The difference in pres- 
sure may be converted by an appropriate factor to volumes per cent CO„ or to meq, per 
liter C0 2 . 

Reagents : 

As under the volumetric method. 
NaOH 1 N (deae rated) 
Prepare as follows: 

1. Allow 25 ml. of solution to run into the chamber. Seal the stopcocks with 
mercury and evacuate to the 60 ml. mark. 

2. Shake for 3 minutes and allow the air to escape through the cup by raising 
the mercury reservoir. 

3. Reseal and repeat at least twice more until no measureable amount of 
residual air remains. 

4. Transfer the deaerated NaOH into a storage chamber with stopcock under 
oil and seal the Up of the storage chamber by immersing in mercury. 

Procedure: 

1. The extraction chamber is cleaned by the use of 10-15 ml. of water and 1 ml. 
of 1 N lactic acid shaken under vacuum, and the cleaning fluid is ejected through the 
cup. 

2. A drop of caprylic alcohol 1b drawn Into the capillary above the top stopcock 
avoiding the entrance of air. 

3. Deae ration of the lactic acid : 7.5 ml. of 0. 1 N lactic acid is placed In the, cup 
and drawn into the extraction chamber. The capillary of the cup and the stopcocktare 
then sealed with mercury and the mercury meniscus is lowered to the 50 ml. mark. 
Shake for 3 minutes. The lower stopcock is now opened and the mercury is allowed 

to slowly rise. The mercury reservoir is now raised to the top position and the bottom 
stopcock closed. 

4. Introduction of plasma sample : The top stopcock Is opened totfhe cup and the 
bottom stopcock opened slowly to allow the lactic acid solution to rise to the 6 ml. 
mark on the cup (5 ml. for the blank "c" correction). The bottom stopcock Is then 
closed and the mercury reservoir Is lowered to the rest position. The sample is then 
delivered using a 1 ml. plpet with or without a rubber tip. The level of fluid in the 
cup Is then lowered to exactly 5 ml. by the use of the bottom stopcock and the capillar- 
ies are then sealed with mercury, allowing a few drops of mercury to enter the ex- 
traction chamber. 

5. Extraction of the gas: With the top Btopcock closed and sealed with mercury, 

137 






the mercury meniscus Is lowered to the 50 ml. mark. The bottom stopcock Is cloned 
and the extraction chamber is shaken for 3 minutes. 

6. Reading of the pressure P .. : P^ is read by allowing the solution to rise slowly 
and evenly to the 2 ml. mark. If any bubbles are present they must be allowed to break 
before the reading is made. At the same time the temperature is recorded. To insure 
complete gas extraction the mercury meniscus is again lowered to 50 ml. and the ex- 
traction chamber is shaken for 3 minutes, and the reading P^ is repeated. 

7. Reading of the pressure P p: The solution is slowly lowered to the middle of the 
large bulb of the extraction chamber and approximately 2 ml. of 1 N NaOH (deaerated) 
Is introduced into the cup. The top stopcock is carefully opened, care being taken to 
avoid the introduction of an air bubble. 1 ml. of the deaerated 1 N NaOH is allowed to 
run slowly (30-60 seconds) into the chamber. The capillaries are again sealed with 
mercury allowing a few drops to enter the chamber. Allow to stand for 1 minutes for 
full drainage, then allow the solution to rise slowly and carefully to the 2 ml. mark. 
Read the manometer P 2 . 

8. Determination of the "c" correction factor : Proceed as under procedure 1-3 
but allow the deaerated lactic acid to rise to the 5 ml. mark in the cup, thus leaving 
2.5 ml. of lactic acid in the extraction chamber. The capillaries are sealed with mer- 
cury, and a p£ reading taken with the solution meniscus at 2 ml. 1 ml, of deaerated 

1 N NaOH is added as under Step 7 and a PS reading taken. The difference pjj - f£ « 
"c" F the correction factor. 

Calculation : 

P C 2 = Pi - P 2 - c q 

For the above given solution volumes and for various temperatures, the factors 
are given in the table. 

C0 2 Factor C0 2 Factor 

Temperature °C Vol. % meg. /X Temp. °C Vol. % meg. /I 

15 27.35 12.44 26 25.81 11.75 

16 

17 

18 

19 

20 

21 

22 

23 

24 

25 



27.19 


12.38 


27.04 


12.30 


26.90 


12.23 


26.75 


12.15 


26.62 


12.10 


26.48 


12.04 


26.34 


11.98 


26.20 


11.92 


26.07 


11.86 


25.94 


11.80 






27 


25.69 


11.69 


28 


25.57 


11.64 


29 


25.45 


11.60 


30 


25.33 


11.56 


31 


25.22 


11. 50 


32 


25.11 


11.44 


33 


25.00 


11.38 


34 


24.89 


11.32 



p x (factors/100) = volumes percent C0 2 

COo 

P co x (factor 2 /l00) = meq. C0 2 /llter 

138 






Exam pig: 



El s 348.7 mm. 
P 2 s 146.4 mm. 
c a 0.5 mm. 






Temperature = 24 C 



P C02 = 348.7 - 146.4 - 0.6, 

P co - 201.8 mm. 
2 



26.07: Factor^ = 11.86 



Interpretation : See Acid Base Balance, p. 321. 



Factor , „.,,.. ._ 2 . „.„ 
201.8 x (26.07/100) = 52.6 vol. % CQ 2 
201.8 x (11.86/100) = 23.9meq.C0 2 /llter 






Alternate Procedure (1): 

If 0. 2 ml. of 5 N NaOH Is used to absorb the C0 2 In the air phase. It Is not neces- 
sary to deaerate the alkali since It dissolves only about 10 per cent as much air as the 
1 N NaOH. 

Alternate Procedure (2): 

1. 5 ml. of lactic acid and 1. ml. of plasma may be extracted as outlined In the 
volumetric procedure and P 2 measured with the gas volume at 2. ml. The gas is 
then ejected without loss of fluid, the capillary is resealed with mercury and the 
pressure measured again at 2.0 ml. gas volume. At 20-28°C. the following equation 
applies: 

(Pi - P 2 ) x 0.266 - 8.6 = Vol; % C0 2 







































139 



CARBON MONOXIDE IN BLOOD 
Micro Gasomctrlc Estimation 

References: 

1, Roughton, F.J.W., and Scholander, P. F. , "Micro Gasometric Estimation of 
the Blood Gases," J. Blol.Chem. , 148, 651-63 (1943). 

2. Kaye, Sidney, Handbook of Emergency Toxicology, Charles C. Thomas - Pub- 
lisher, Springfield, Illinois, pp. 134-40 (1954) 

Principle : 

In the microgasometrlc technique for estimating the carbon monoxide In a drop of 
blood, 40 cmm. of blood are mixed In a 1 ml. syringe with ferricyanlde containing po- 
tassium bicarbonate and saponin. An acetate buffer Is then added. The CO which is 
evolved upon shaking provides a gaB phase for the extraction of the other gases of the 
blood and reagents; i.e. , 0„, CO, and N 2 . After the extraction Is complete, the CO„ 
and the extracted 0„ are absorbed by alkaline pyrogallol; the small bubble which 
remains is measured in a graduated capillary attached to the nozzle of the syringe. 
The CO Is absorbed by Winkler's solution and the gas bubble measured again. From 
the difference between the two readings the CO content of the blood may be calculated. 
No blank correction is required for the reagents since the blood Itself Is the only 
source of CO. 

Apparatus ; 

A special syringe analyzer and pipets are required. These are described in the 
original reference. They maybe obtained by writing James D. Graham, 11 MontweU 
Avenue, Haddonfleld, New Jersey. 

Syringe, luer, 2 ml. and 10 ml. 

Needle, hypodermic, 21-gage. 

Reagents : 

1. Distilled water. 

2. Caprylic alcohol. 

3. Ferricyanlde solution. 2.5 grams of K„Fe(CN) 6 , 0.6 grams of KHCO„, and 
0. 1 grams of saponin, are ground In a mortar and dissolved in water to make up to 10 
ml. This solution should be renewed every three days. 

4. Acetate Buffer. 70 grams of sodium acetate {NaC 2 H 3 02°3H 2 0) are dissolved 
In 100 grams of water and 15 mis. of glacial acetic acid then added, 

5. Urea. Dissolve 45 grams of urea in water and make up to 100 mis. 

6. Winkler's Solutioh. 20 grams of cuprous chloride (CuCl). 25 grams of ammo- 
nium chloride, and 75 grams of water are placed in a bottle just large enough to con- 
tain them. The bottle is corked, shaken with as little air as possible, and the precipi- 
tate allowed to settle. A coil of copper wire is placed in the solution which is then 
covered with a layer of paraffin oil. After some time the reagent becomes colorless. 

7. Pyrogallol Solution. 15.0 grams of powdered pyrogallol (CgH 3 (OH) 3 ) are added 
to 100 mis. of 20 per cent NaOH in a rubber stoppered bottle, and covered with a layer 
of paraffin oil. The pyrogallol is dissolved under the oil by stirring with a glass rod. 

140 



< 



< 









- mm 

_ I 




Figure a. Transfer of Blood from 
pfpet to capillary . 




Figttr* b. Shaking of syringe 
and extraction of gases. 




Figure c, Absorption of 
CO f by alkaline reagent. 




Figure d, Absorption of COg 
by Winkler's solution. 



These ill utt rat loos have been adapted front Reference 1. 

Figure M. Carbon awaoKide analysis. 



140n 






Blo_odSa.mplea : 

A bloort apucimen of approximately 6 ml. la collected by sterile technique, with 
a minimum of exposure to Hie air during «li stepu of fho collection. The specimen 
should be drawn and transferred to the container u lowly to prevent aeration. It la 
then placed In a clean screw-cup tost tube, containing 5 mg. of lithium oxalate. Over- 
lay the specimen with 1 ml. of Kiiold petrolatum and gently stir with a clean rod to 
effect solution of the oxalate. Fill the test txbo v/Uh liquid petrolatum and screw on 
the cap. 

Procedure : 

1. The syringe Is held vertically and any liquid in the cup of the syringe is re- 
moved by vacuum suction. The ferrlcyanlde solution is drawn to the bottom of the 
syringe and expelled through the cup and removed. This procedure is repeated twice 
with fresh lots of ferrlcyanlde, the dead space finally being left full of ferrlcyanlde 
without trapping any air bubbles. No grease or oil is used in the syringe. 

2. The glass cup is filled to the mark with ferrlcyanlde and the latter is drawn 
down to the bottom of the cup. 

3. A drop of caprylic alcohol is deposited on the bottom of the cup. 

4. The pipet Is filled to the mark with blood, wiped, and held at a slight angle to 
the horizontal so that the blood does not run out when both ends of the pipet are open to 
the air. With the syringe at the same angle, the pipet is cautiously introduced into the 
glass cup, and its tip pressed snugly, but not too vigorously, against the bottom of 
the cup. (Fig. 24a, p. 140a). 

5. By pulling out the plunger gradually the blood is slowly and evenly drawn Into 
the capillary, followed by a bubble of air about 1 mm. in length. If the tip Is properly 
ground, and the right amount of pressure is applied, no appreciable caprylic alcohol 
is drawn in during this step. The bubble of air prevents any blood being sucked back 
Into the tip when the pipet Is removed. 

6. The pipet Is quickly removed and the bubble of air is then expelled through the 
caprylic alcohol, with the aid, if necessary, of a piece of fine wire or by tapping the 
capillary. 

7. A trace of caprylic alcohol, I.e. , about two division lengths of the capillary, 
is drawn onto the top of the blood and the rest of the caprylic alcohol 1b removed from 
the cup by suction, 

8. The cup Is filled to the mark with acetate buffer and the latter drawn down to 
the bottom of the cup. 

9. The cup Is then immediately filled to the top with 45 per cent urea, and then 
closed firmly with the finger. 

10. The closed apparatus Is vigorously shaken in the horizontal position, the plund- 
er being gradually drawn out as the CO, and the other gases are evolved, the gas pros - 
sure being kept roughly at atmospheric. (Fig. 24b.) The tot.il volume evolved oho old I : 
about 0, 75 cc. If the amount la appreciably more or less than this the concentration 

of KHCOg in the ferrlcyanlde reagent should be correspondingly adjusted. Shaking Ib 
continued for a total of 2 minutes. 

11. The finger is cautiously released, the syringe plunger being maniuplated so as 
to keep the gas meniscus in the capillary. A bwwII ,'inw - «f urea Is run down Into 

141 



the capillary and left there until the walls are perfectly clean. 

12. Three-quarters of the urea tn the glass cup Is removed, and the rubber cup 
adjusted and filled with pyrogallol solution without trapping air bubbles. 

13. A little pyrogallol is drawn into the syringe. This absorbs some CO„ and 0„, 
causing a partial vacuum which quickly sucks in more pyrogallol until only a small 
bubble consisting of N 2 and CO (if any was originally present in the blood) Is left at 
the top of the syringe. (Fig. 24c.) The absorption takes a few seconds and just before it 
is complete the residual bubble is screwed slowly and carefully up into the capillary 
by manipulation of the plunger. 

14. The rubber cup Is removed, and the glass cup is emptied by suction. 

15. The capillary Is placed for half a minute In a beaker of water at room temper- 
ature. 

16. It Is then removed, dried by light wiping, care being taken that the capillary 
Is not handled, and the volume of the bubble read, V. divisions. 

17. The glass cup Is flushed clean with water and left filled. About three-quarters 
of this water is pulled quickly down into the syringe, forming a layer on top of the heav- 
ier blood mixture, The bubble, with clean water below it, is then at once run up to the 
top of the capillary. 

18. The glass cup Is emptied of water and filled with Winkler's solution. 

18. The syringe is then pointed with the cup downwards, the capillary making a 
slight angle with the horizontal. By cautiously screwing in the plunger the gas bubble 
is driven out into the glass cup where it rests near the junction of the capillary and the 
cup. (Fig. 24d.) As soon as the bubble is free in the cup, Winkler's solution Is sucked 
behind it so as to half fill the capillary. Gentle rotation for a few seconds completes 
the absorption of the CO. The syringe Is then turned to the vertical position with the 
cup downwards and the gas bubble is sucked back into the capillary and Its volume, V2, 
measured as above. 

20. To wash the instrument, the plunger Is pulled out under a stream of running 
water, and the blood mixture poured out. The syringe is filled and emptied several 
times with water before the plunger is restored. The plunger should never be forced. 
If there is resistance due to precipitates inside it the whole syringe should be re- 
cleaned. Occasional cleaning with chromic acid is recommended. 

Calculations : 

The carbon monoxide content of the blood In volume per cent equals (Vj - V$ x f, 
where f is the correction factor for temperature, aqueous vapor pressure, and baro- 
metric pressure. In COHb estimations at saturations below 8 volumes per cent, at 
room temperature and at sea level it Is often In practice accurate enough to let f = 0. 9. 

Example: 

Reading of V*! 5. 2 

Reading of V 2 4. 7 

0.9(5.2* - 4.7) = volumes per cent carbon monoxide 
0.9(0.7) s 0.61) ■ volumes per cent carbon monoxide 



( 



( 






142 






At the U. S. Nav:il Medical School, further calculations are made as folic va: 
The hemoglobin of the blood Is determined by any convenient method, and this 
value Is multiplied by 1.36 {conversion factor - one gram hemoglobin equals 1.36 
volumes per cent carbon monoxide) to obtain the carbon monoxide capacity of the blood. 
The carbon monoxide content multiplied by one hundred, then divided by the carbon 
monoxide capacity gives the percentage of saturation of the blood specimen. 

Example : 

Hemoglobin - 14 grams 

Carbon monoxide content — 0.63 volume per cent 

Carbon monoxide capacity — 14 x 1.36 - 19,0 

Carbon monoxide saturation = P , : , ? . 3 , . £ _ * 2 P . -3,3 per cent saturation 

19 

Notes : 

1. One objection to this method Is that the alkaline pyrogallol solutions used may 
evolve traces of CO. This tendency diminishes with the strength of alkali and pyrogallol 
used, but increases with the partial pressure of 2 and the time of contact of the pyro- 
gallol solution with the gas phase. The CO Is thus apparently a secondary product of 
the reaction between On and pyrogallol. With the amounts of 0_ yielded by 40 c. mm. of 
blood this pyrogallol solution has not been found to evolve any CO when used In the 
syringe technique. 

2. The alkaline pyrogallol solution must be removed Into the syringe from under 
oil without exposure to air. 

3. A drop of caprylic alcohol Is introduced between the barrel and plunger of the 
syringes used for the reagents, to lessen the exposure to air, and also to prevent 
freezing of syringe due to the drying of some of the reagents. 

Interpretation : 

The normal carbon monoxide blood level is to 2 per cent for non-smokers and 
1 to 3 for smokers. The minimum lethal dose is a 40 per cent car boxy-hemoglobin 
saturation. If death is due to carbon monoxide, samples drawn from the body weeks 
later will still so Indicate since putrefaction will not affect the carbon monoxide level. 

For a blood sample of 40 c. mm as described In the technique the accuracy of a 
single determination is 0.15 to 0.20 volume per cent. 















143 



CEPHALIN FLOCCULATION 
Serum 

R eference : 

Hanger, F. M. , Jour. Clin. Invest. -18, 261 (1£>39). Am. J. Med. Scl. 202 , 
48 (1841), 

Pr inciple : 

Disturbances of the liver parenchymal cells may be detected by noting the capac- 
ity of blood serum to flocculate a colloidal suspension of cephalln and cholesterol. 

Reagents : 

1. Cephalln-cholesterol mixture. This consists of 100 mg. of partially oxidized 
cephalln and 300 mg. of cholesterol. The mixture Is purchased from suppliers such 
as Dlfco Laboratories. Prepare the ether mixture by adding 6 ml. of anesthetic ether 
per unit to effect solution of the contents. If turbidity persists, add one drop of dis- 
tilled water to obtain a clear solution. This solution constitutes the stock ether antigen 
of Hanger and Is stable for months if kept tightly stoppered to prevent evaporation. 
Keep refrigerated. 

2. Preparation of test antigen from the cephalln-cholesterol mixture. Add slowly 
with stirring, 0.5 ml. of stock ether-antigen to 18 ml. of distilled water at 66-70° C. 
Then heat slowly to boiling. Simmer until the volume is reduced to 16 ml. This should 
result in a stable, milky emulsion. Prepare fresh each day. 

Procedure : 

Place 1 ml. emulsion in a test tube containing 0, 2 ml. of the patient's serum (not 
over 24 hours old) and 4 ml. 0.86% saline. Mix well by shaking. 

Control tube - 4 ml. saline / 1 ml. emulsion. If possible run positive and negative 
control sera. Read after 24 and 48 hours. Record results. 

Interpretation: 

24 hours = negative 

48 hours = negative to 2 / 

A negative result is of value In excluding parenchymal hepatic disease. Some 
workers believe that a positive result is due to various combinations of three conditions: 

1. Increase of gamma globulin 

2. Decrease of serum albumin 

3. Change In properties of serum albumin. 



( 



< 



negative 


= milky solution - no visible flocculatlon 




1/ 


= milky notation - faint flocculatlon - dispersed 




2/ 


■ milky solution - definite flocculatlon - dispersed 




3/ 


- milky solution - partlcally settled flocculatlon 




4/ 


- clear solution - complete flocculatlon 






144 


C 



CHLORIDES 
Serum, Spinal Fluid, and Urine 

References; 

1. Shales, O. , and Shales, S.S. , J. Biol. Che in. , 140 , 879 (1941) 

2. Franco, V, , and Klein, B. , J. Lab. Clin. Med. , 37, 950 (1961) 

3. Salfer, A. and Hughes, J., J. Biol. Chem 129, 273 (1939) 

4. Whltehorn, J.C. , J. Biol. Chem. , 45, 449 (1921) 

5. Wilson, D.W. , and Ball, E.G., J. Biol. Chem., 79, 221 (1928) 

6. King, E. J. , and Bain, D.S. , Blochem. J. , 48, 51 (1961) 

Method I. (Reference 1) 

Principle : 

This is a mercurlmetric procedure. As with silver ions, chloride also combines 
with mercuric ions according to the following equation: 

2 Cl~ / Hg^ HgCl 2 (unionized) 

No precipitate is formed, contrary to the effect of silver ions forming Insoluble silver 
chloride but the mercuric chloride is only very slightly dissociated into Ions, and the 
end point Is recognized by the appearance of Hgrr i ons in solution. The recognition of 
Hgrr is made possible by various Indicators such as urea, sodium nitroprusside, etc. , 
but in thia method use is made of s-diphenylcarbazone (EK Co. #4459) which changes 
from colorless or light yellow to an Intense blue when combined with Hg^V. It is essen- 
tial that the pH at the end point be between 1 and 2. 

Apparatus : 

1. Flasks 25 ml. Erlenmeyer. 

2. Mlcroburet calibrated in 0. 01-0.005 ml. 100 drops should equal 1 ml. 

Reagents : 

1. Mercuric nitrate solution 1. 6-1. 8 g. Hg(N0 3 )„ are dissolved In a few hundred 
ml. of water with the addition of 20 ml. of 2 N HNO3. Make up to 1000 ml. with water. 

2. Standard sodium chloride solution. 

Sodium chloride A.R. Is dried at 120° C. and 584.5 mg. are dissolved in water 
and made up to 1000 ml. This solution contains 0.01 mllllequlvalents of chloride/ml. 
It is used for the standardization of the mercuric nitrate solution each day. 

3. Indicator — 100 mg. of diphenylcarbazone (EK Co. #4459) Is dissolved In 100 ml. 
of 95% alcohol and stored in the dark, preferably refrigerated. If the end point becomes 
sluggish, discard the solution; it Is better to prepare new solution monthly. (It has 
been suggested that a sharper endpbint is achieved by using dlphenylcarbazide-sulfonic 
uciu. f 

The stability of the diphenylcarbazone solution is improved by the use of a product 
obtained by recrystalllzation as follows: 

Saturate 200 ml. of 95% ethyl alcohol with the commercial s-diphenylcarbazone. 
Filter or decant the solution. Add water to the solution gradually until the maximum 
turbidity Is obtained. Allow to stand at refrigerator temperature overnight. Collect 
the crystals by filtration using aBuchner funnel and a hard (Whatman #50) filter paper. 
Dry in a desiccator, do not use heat. Store the solid In the refrigerator. 

145 



Procedure: 

1. Whole serum: To 2.0 ml. of water In a 25 ml. Erlenmeyer flask Is added 
0.20 ml. of serum by 8 times rinsing from a 0.200 ml. "to contain" plpet, and 0.06 
ml. of Indicator (4 drops). The color In this slightly turbid mixture la salmon ru$i, 
which changes to a deep violet, and then to a light yellow, as titration with the acid 
mercuric nitrate solution proceeds and at the end-point a change to a pale violet occurs, 
which can be seen quite easily. The drops from the buret should be very small, about 
100 to the ml. 

2. Serum filtrates: To 2.0 ml. of filtrate ( = 0.2 ml. serum) in a 25 ml. Erlen- 
meyer flask is added 4 drops of indicator solution and the titration carried out as noted 
above. The color changes are somewhat different but at the end point changes to a 
bright blue. 

Calculation : 

T g = ml. mercuric nitrate solution required for titration of 2.0 ml, of standard 
sodium chloride solution ( = 0.02 meq.). T u > ml. mercuric nitrate reagent required 
to titrate 0.2 ml. of serum (or equivalent filtrate). 

C 8 = meq, CI" in 2.0 ml, of standard NaCl a 0,02 

(T u /T s ) x C 8 a meq. Cl~in0.2ml. serum 

(T u /T s ) x C s x ~~ - meq . CI" Alter serum - (T U /T B ) x 100 

or (T u /t s )C s x « x 35.5 a mg.% (as chloride Ion) = <T u /tg) x 355 

(T u /T fl ) x 585 = mg.% as NaCl 

Note : In order to increase the volumes measured In titration, we actually use 
5 ml. of a 1:10 filtrate of serum (equivalent to 0.5 ml. of serum) and 5 ml. of the 
standard containing 0.05 meq. CI". 

For urine and spinal fluid using a 0. 5 ml. samples and the T of 2 ml. of standard, 

CT../T ) x 4 = meq. Cl"/100 ml. sample 

i ir g' 

<T U /T B ) x 40 = meq. Cr/liter 

(T /T ) x 142 a mg. CI". 100 ml. 

(T u /T s ) x 234 = mg. NaCl/100 ml. 

N otes : 

The authors and others state that direct titration of serum gives results 1-3 meq. / 
liter higher than the filtrates and ascribe this effect to the adsorption of Cl" by the pre- 
cipitate. However, the protein present in serum does affect the titration and filtrates 
should be used , for the greatest precision. 

146 



( 



( 






1. Hypodermic needles as buret tips arc not satisfactory since the mercuric Ion 
reacts with the metal In the tip. 

2. In preparing the mercuric nitrate solution the amount of nitric acid specified 
should be used; otherwise the end point will not be sharp. 

3. Urine and Spinal Fluid: To 0,5 ml. urine or spinal fluid add 5.0 ml. 0.05 N 
HK0 3 and titrate as above. Calculation as above except taking into consideration the 
volume of urine or spinal fluid used. 

Chloride Method II. (Reference 4) 

Principle : 

The chloride Is precipitated by the addition of a measured amount of silver nitrate. 
The excess silver is then titrated with NH.CNS. The indicator used is ferric ammo- 
nium sulfate, which with excess thiocyanate gives the red color of Fe(CNS) 3 . 

Reagents : 

1. Silver nitrate - 5.813 g. AgNO diluted to 1 liter with HgO (distilled). 
Preserve In brown bottle. 1 ml. = 2.0 mg. NaCl. 

2. Ammonium thiocyanate - Dissolve 3 g. NH.SCN in 1 liter distilled HO. 
Standardize against AgNO. , using the technique given under procedure and 
adjust so that 5 ml. - 5 mi. AgNO,. 

3. Ferric ammonium sulfate - solid. 

4. HNO3 concentrated. 

Procedure : 
Serum: 

1. To 10. 00 ml. of a tungstic acid filtrate of serum, add with stirring 5. 00 ml. of 
silver nitrate solution. Add 4 ml. concentrated HKO„, mix and allow to stand for five 
minutes In the dark. 

2. Add a few grains (about 0.3 g.) of ferric ammonium sulfate and titrate to the 
first reddish-brown endpoln' lasting ten to fifteen seconds. 

Spinal Fluid: 

1. To 1.00 ml. (or 0.5 ml.) spinal fluid, add 9 ml. HO and 5.00 ml. AgNO. 
solution, mixing during the addition. Add 4 mi. concentrated KNO„, mix, add a 
few grains of solid ferric ammonium sulfate. 

2. Allow to stand In the dark 5 minutes. 

3. Titrate with ammonium thiocyanate solution to the first lasting reddish brown 
color. 

Urine : 

1. Dilute urine 1:10 (1 ml. /9 ml. H 2 0). 

2. To 10.0 ml. of diluted urine add 5. 00 ml. AgNO„ and proceed as above under 
spinal fluid chloride. 

Calculations : 

Jf £ is the volume In ml. of NH.SCN used in the back-tltration: 
Then (5 - t) x 2 = mg. NaCl in the sample. 

147 



(6-t) x 2/58. 5 a mcq, CI" in the sample and (5-t) x 2 x 35. 6/58. 5 - 
mg Cl~/sample 

(5-t) x 2/58.5 x 1000/0.5 u meq, Cl~/Hter (using a 0.5 ml. sample) 

Chloride Method m. (References 2 and 3) 

Principle : 

Serum, spinal fluid, and urines are deproteinized by the addition of the reagents 
of Somogyl (1945) ZnS0 4 and Ba(OH)„ (see deprotelnizlng methods p. 79) and the filtrate 
is titrated with silver nitrate using dlchlorofluoroscein as an adsorption Indicator. 
The precipitate AgCl with the first excess of Ag^ becomes an ion, (AgClAg»*) which 
strongly adsorbs the indicator dichlorofluoresceln, (yellow in solution) which turns to 
red on the solid phase. 

Reagents : 

1. Ba(OH) 0. 086 N. Prepare by dilution of 0. 3 N Ba(OH) described under Method 
IV of deprotelnizlng methods, (add 2 ml. to 6 ml. of water). The Ba(OH) is diluted at 
the time of use and kept protected from the C0 2 of the air. 

2. ZnSO., 5% - described in deprotelnizlng methods. 

3. Standard NaCl - 1. 000 g. dried reagent NaCl is dissolved up to 1000 ml. with 
water; 1 ml. = 1. mg. NaCl. 

4. Silver nitrate 0. 1 N.. Weigh out exactly 8. 4945 g. reagent grade silver nitrate, 
transfer to a 500 ml. volumetric flask and dilute to 500 ml. Keep in a brown bottle in 
the dark. This may serve as the primary standard Instead of the NaCl. 

5. Silver Nitrate . 02 N. Prepared by dilution of the 0. 1 N solution and standard- 
ized against NaCl, using the technique described for blood filtrates under procedure. 

6. Dlchlorofluoroscein. 0.05% in 70% ethyl alcohol. Weigh out (analytical balance) 
50 mg. £ one mg. of dlchlorofluoroscein (EKCo. #373) dissolved In a mixture of 

74 ml. of 95% ethyl alcohol and sufficient water to give 100 ml. (graduate). 

Procedure : 

1. Serum: 

Add 7.0 ml. dilute Bt(OH)„ solution to 1.0 ml. of clear serum; then add 2,0 ml. 
of ZnSO^ solution, stopper and mix well by shaking. Centrifuge to pack precipitate well. 
Transfer 2.0 ml. aliquots to 25 ml. Erlenmeyer flasks; add 1 drop of Indicator and 
t\ . ite with 0.02 N AgN0 3 solution until the first pink is seen through the entire solution. 

2. Spinal Fluid: 

0.5 ml. of clear spinal fluid Is treated with 3.5 ml. of dilute Ba(OH) solution 
and 1.0 ml. ZnSO. solution, 2.0 ml, aliquots are titrated as described above. 

3. Urine : 

To 0. 2 ml. urine in a pyrex test tube are added 3 drops of 3% HgO™ (chloride 
free). Heat tube in a boiling water bath for two minutes. Cool. Pipet into this tube 
3.5 ml. dilute Ba(OH) ; 1.0 ml. ZnS0 4 and 2.0 ml. HgO. Mix well. Centrifuge and 
remove 3.0 ml. aliquots. 

148 



,j.ih illation : 

T Ag = ml. Silver Nltratc(b. 02 N) 

Serum and Spinal Fluid; 

T^g x 0.02 = meq. Cl"/sample: meq x 35.5 i mg, Cl~ 

meq x 58.5 = mg. NaCl 

T Ag x 0.02 x 1000/0.2 = meq Cr/Uter of serum 

T Ag x 100 x meq Cl~Alter 
Urine: 

T._ x 0.02 x 6.85/3.00 x 1000/0.2 meq. Cl"/llter of urine 

AS 

T A x 228.3 = meq Cl"/Uter of urine 

Chlorides Method IV. (Reference 6) 

Principle : 

Silver lodate (insoluble) reacts with chloride in solution to form a precipitate of 
sliver chloride and leaves lodate In solution. NaCl / AglOg — > AgCl / NaI0 3 . The 
iodate in solution which is stoichlometrlcally equivalent to the chloride in the sample 
used, is determined by adding potassium iodide in acid solution and titrating the lib- 
erated iodine with thlosulfate. For reagents, procedure, and calculations, see the 
original reference. 

Interpretation : 

See Electrolyte Balance In Appendix. 












149 



SWEAT ELECTROLYTES 

No special preparation of the patient is necessary. Wash the mid-back with dis- 
tilled water and dry. Do not touch this area with your fingers. With forceps place a 
three inch square gauze pad (Curity brand) In a stoppered Erlenmeyer flask and weigh 
on an analytical balance. Still using forceps, place the pad on the back and cover with 
a slightly larger piece of plastic sheeting. Tape the sheeting to the back with eight 
strips of one Inch adhesive tape - two strips of tape to each side of the sheeting. Place 
the patient In a plastic suit which has an elastic neck and a zipper in the front. Cover 
patient with one or two blankets. Note the time. At intervals note the amount of sweat 
condensed in the bag and if it appears sufficient remove patient from bag. The average 
collection time is 60 minutes (range - 15 minutes to 90 minutes). Remove the plastic 
sheeting. Then replace the sweat soaked gauze pad (use forceps) into the same stop- 
pered ErlenmevT flask and reweigh. The difference between the two weights, the wet 
minus the dry, represents the amount of sweat collected. 

Procedure : 

Add 20 ml. of distilled water to the flask containing the sweat-soaked gauze pad. 
Allow the sweat and distilled water to become well mixed. Now place S ml. of this 
diluted sweat sample in an evaporating dish. Add 10 drops of diphenylcarbazone indi- 
cator. Titrate to end point with standardized mercuric nitrate solution In the usual 
manner. 



Sweat Chl oride 
Calculation: 



If s is the weight of sweat collected, then: 
20 ml. / b _ dilution factor (F d ) 









Sweat CI" (meq. /L) = (T u /T fl ) x C s x ^| 



Examp le: 



Flask stopper, pad and sweat 53. 2239 g. 

Flask stopper and dry pad 51.7320 g . 

Sweat (s) 1.4919 g. 

20 / B _ 21.4919 = u 4 

s 1.4919 d 

Using 5 ml. of standard chloride and 5 ml. of diluted sweat: (cont.) 



150 



( 



( 






T„ = 4.988 ml. 

T u s 0.63 ml. 

C_ - 0.05 meq. CI" 



Sweater = (0.63/4.988) x 0.05 x i°-21 . meq./l. 

6/14.4 

= 0.1263 x 0.0& X2880 
= 18. 2 meq. Alter 

References : 

1. Darling, R.C.: Am. J. Med. Sc. , 225, 67 (1953). 

2. di Sant'Agnese. P. A.: Pediatrics, 12, 549 (1953). 

3. Schwachman, H. : American Academy of Pediatrics, Oct. 5, 1954, Chicago. 

4. Schwachman, H. : Advances In Pediatrics, Vol. VII, 249-323, 1955. 

Mucoviscidosis accounts for almost all cases of pancreatic insufficiency in chil- 
dren. The incidence in the population of the United States is between 1 in 600 and 1 in 
10,000 live births, with a probable average Incidence of 1 in 2,500. It is a familial 
disease displaying the characteristics of a mendelian recessive gene. Both parents 
must be carriers of the trait. In an affected family, the disease may occur In approx- 
imately 25% of the offspring, and two-thirds of the non-affected children may be 
carriers. Most patients with this disease have manifestations before 6 or 8 months 
of age. 

The measurement of the sodium and chloride concentration in body sweat is a 
well established diagnostic procedure In the study of patients suspected of having 
Mucoviscidosis (cystic fibrosis of the pancreas). The salt concentration runs two to 
four times as high as that seen In a control group. This change is independent of the 
pulmonary or pancreatic process, and has no association with renal, adrenal, or 
gastrointestinal tract dysfunction. The excessive salt loss readily explains the sb- 
normally high incidence of heat prostration in children with mucoviscidosis. 

A value of sodium above 80 meq. /L of sweat or a value of chloride above TO meq. /L 
of sweat Is fkipacterlstlc of the disease. A small percentage of parents and apparently 
well siblings i>f affected children will show such an elevation. The absence of percep- 
tible sweating in the neonatal period makes this test invalid during the first three to 
five weeks of life. 

Potassium content of sweat In patients with mucoviscidosis is alsc increased but 
is not sufficiently abnormal to be of any diagnostic value. 



151 



CHOLESTEROL AND ESTERS 

Serum 

Reference B: 

1. Bloor, W.R. and Knudson, A., J. Biol. Chem. , 27, 107 (1916) 

2. Bloor, W.R. and Pelhan, K.F. , Allen. D.M. , J. Biol. Chem., 62, 191 (1922) 

3. Shoenhelmer, R. , and Sperry, W.M. , J. Biol. Chem. , 106. 745 (1934) 

4. Sperry, W. M. and Webb, M. , J. Biol. Chem. , 187, 97 (1960) 
6. Carr, J. J. , aad Drekter, I. J. , Clin. Chem. , 2, 963 (1966) 

Method 1 (Reference II 

Principle ; 

Serum is added to a mixture of alcohol and ether, which extract* the lipid* and 
precipitates the protein. 

Total Cholesterol 

An aliquot of the alcohol-ether extract Is evaporated to dryness, the cholesterol 
In the residue Is extracted with chloroform, and color 1* developed by the Liebermann- 
Bu re hard reaction, Equimolecular amounts of free and ester cholesterol give Identical 
amounts of color if the determination Is made at 25°C. and the color is read at 46 min- 
ute*. 

Cholesterol Esters 

Dig! ton in Is added to the alcohol-ether extract of serum, precipitating free 
cholesterol as the digitonide. The dlgltonlde Is Insoluble in petroleum ether and re- 
mains behind while the esters are extracted with this solvent. The petroleum ether 
Is then evaporated to dryness. The cholesterol esters in this residue are extracted 
into chloroform and the color developed as above. 

Reagents : 

The solvents mentioned may be satisfactory for use without distillation. This may 
be determined by repeated experimental determination of the slope and shape of the 
standard density vs. concentration (D/C) curve, and should include a density vs. time 
curve also. If a solvent requires purification, It should be redistilled. 

1. Alcohol- (ethyl) 95%. 

2. Ether-(diethylether). 

3. Alcohol-ether, Three parts of 95% alcohol plus 1 part of ether (by volume). 

4. Chloroform - may be used without redistillation but should be stored over an- 
hydrous sodium carbonate to keep it dry and neutral in reaction. 

6. Acetic anhydride reagent, hi a dry container, place 10 ml. of glacial acetic 
acid, and 20 ml. acetic anhydride. Cool well in an ice water bath and add 2 ml. of 
ice cold concentrated sulfuric acid. Mix well. Allow to warm to room temperature 
before use. 

6. Cholesterol standard solutions: Dissolve 40.0 mgm. cholesterol up to 500 ml. 
with alcohol-ether mixture . Keep tightly stoppered In a glass stoppered bottle. Pre- 
pare new standard monthly. 10 ml. = 0.8 rag. of cholesterol. 



152 



(. 






7. Petroleum ether (hcc page 157). 

8. Digitonin - 1% solution In 95% ethyl alcohol. Digitonin dissolves slowly and may 
require heating in a water bath. 

Procedure : 

Note : All apparatus must be dry. 

Preparation of protein-free lipid filtrate; In a 50-ml. volumetric flask place about 
25 ml. alcohol-ether mixture. Add slowly with shaking, 2.0 ml, serum. Heat mixture 
to boiling in a waterbath, (no flame). Cool to room temperature. Dilute to volume with 
alcohol-ether. Mix. Filter through Whatman #43 filter paper (or other fat-free filter 
paper), into a glass-stoppered bottle, avoiding evaporation of solvent. 

Total Cholesterol: 



1. Transfer 10. ml. of filtrate to a 100 ml. beaker. Evaporate Jtrst to dryness, 
using a gentle (I) air current if desired, dry under Infra-red lamp 10 minutes. 

2. Extract the residue, first with 3 ml. then with 2 ml. four times with chloro- 
form (3,2,2,2,2,). Each time rinse down the sides, cover with a watch glass, boil 
until a drop of condensed CHClg drops from the watch glass, using a hot plate set at 
gentle heat (or a waterbath). 

3. Filter each extract through a funnel with a bit of fat-free cotton in the stem, 
into a glass stoppered test tube graduated at 10 i ml, 

4. Cool and dilute carefully to volume with chloroform. 

5. Carry 10.0 ml. of the cholesterol standard through the determination in the 
same way. 

6. Immerse standard and unknown cholesterol solutions In a water bath at 25°C. 
for 10 minutes. 

7. Add to each, 3.0 ml. of acetic anhydride -sulfuric acid reagent, stopper and 
mix. Replace In water bath and read at exactly 45 minutes after the addition of the re- 
agent. 

8. Set the photometer at 100 with chloroform (not water). Transfer the colored 
solution to a photometer tube and read using filter 640 (or 620) or a spectrophotometer 
set at 625 mu. (Care should be take- to avoid Introduction of water.) 

Cholesterol Eaters : 

1. Into a 50 ml. beaker, place abouL 10 mg. of digitonin and dissolve with heat- 
ing In 1-2 ml. of alcohol, (or use 1 ml. of 1.0% alcoholic digitonin). 

2. Add with a pipet 15 ml. of alcohol-ether extract of serum (prepared as above). 
Mix with the digitonin solution. Evaporate just to dryness using a gentle air current 
and gentle heat. Do not, overheat . Place the flask under the infra-red lamp for ten min- 
utes. 

3. Add 15 ml. petroleum ether, cover with a watch glass. Boil on hot plate. (No 
flames nearl No sparking contact!) until 1 > a ii' the volume remains. Pour through cotton 
Into a 100 ml. beaker as in total cholesterol and re- extract with 8 ml. , boil to 1/2 
volume and filter, repeat with 6 ml. then villi 4 ml, and again with 4 ml. (16,8,6,4,4) 
Some workers prefer using (15, 12, 10,8,6), 

153 



4. Evaporate the extracts Just to dryness, heat under lnfra-rcd lamp 10 min. 
and proceed as for total cholesterol, extracting repeatedly, using chloroform (3, 2, 
2, 2, 2). 

5. Proceed with dilution, cooling, color development, etc. as In total choles- 
terol using the same standard. 

Calculation : 

Total Cholesterol: Pu K <j.8 x 15P. H ... . A#w . , 

D * w "° 04 s m g- cholesterol/100 ml. serum. 



or 



5u x200 . mg.% or f^ x D tt * mg.% 
s ■ 



Cholesterol Esters: n „ _ 100 L i * i /««» • 

=u x 0.8 x -^ = mg. cholesterol esters/100 ml. serum 
D fl 0.6 

-uxi33 - mg.% cholesterol esters. 

■ 

Interpretation; 

Normal values of total cholesterol range from 160-260 mg.%. Normal esters are 
60-75% of total. 

Total cholesterol Is Increased In diabetes mellltus with Ilpemla, nephritis, obstruc- 
tion of bile ducts. % ester decreases in liver diseases. In thyroid diseases cholesterol 
varies Inversely with the B.M.R. 

Method n (References 3 and 4) 

Principle : 

Serum proteins are precipitated and lipids are extracted by means of an alcohol- 
acetone mixture. Free cholesterol Is precipitated by the use of digltonin without sapon- 
ification and the total cholesterol is precipitated by digltonin after saponification. The 
separated and washed digitonides are then determined by the Liebermann-Burchard 
reaction, after being dissolved in glacial acetic acid. 

Reagents : 

1. Acetone -alcohol mixture. Mix equal volumes of acetone and 96% ethyl alcohol. 

2. Acetone-ether mixture. Mix 1 volume of acetone with 2 volumes of ether (anes- 
thesia ether may be used). 

3. EthyL ether-analytical reagent or anesthesia ether may be used. 

4. Glacial acetic acid. 

6. Digltonin, 0. 5% solution In 50% ethanol. Dissolve 500 mg. of digltonin in 100 ml. 
of the alcohol at 60° C. 50% ethanol is prepared by mixing 55 ml. of 95% alcohol and 
45 ml. of distilled water. 

154 






6. Potassium hydroxide 33%. Dissolve 10 g. KOH in 20 ml. distilled water. Store 
in a bottle with a dropping pipot with rubber bulb. 

7. Phcnolphthalein. 1% in 95% cthanol. 

8. Acetic acid 10%. Dilute 10 ml. of glacial acetic acid to 100 ml. with water. 

9. Concentrated sulfuric acid-analytical grade. 

10. Acetic anhydride. 

11. Acetic anhydride -sulfuric acid reagent. Place an amount of acetic anhydride 
sufficient for the number of samples to be read in a glass -stoppered flask and chill la 
an ice bath. With the flask still In the Ice bath, add concentrated sulfuric acid in the 
proportion of 1 ml. to 20 ml. of the acetic anhydride with agitation. Insert the stopper, 
and remove the flask from the bath, shake vigorously for a few moments and return to 
the Ice bath. 

12. Cholesterol stock standard. Dissolve 100 mg. (analytical balance) of cholesterol 
in glacial acetic acid and make up to 100 ml. with acid. 

13. Cholesterol working standard. Dilute 10 ml. of the stock standard with glacial 
acetic acid up to 100 ml. This contains 0. 1 mg. cholesterol per ml. of solution. 

Procedure : 

Lipid Extraction and Deprotelnlzatlon. 

1. Add 1.00 ml. of serum, plasma, or blood , dropwlse, with constant shaking, to 
about 10 ml. of alcohol -ace tone mixture in a 25-ml. volumetric flask, glass -stoppered. 

2. Heat carefully In a boiling water bath, with shaking, Just to the boiling point. 
Remove from the boiling water bath, but continue shaking for 30 to 60 seconds. 

3. Cool to room temperature, make up to volume with alcohol-acetone mixture. 
Insert the stopper and mix thoroughly. 

4. Filter through a fat-free filter paper (Whatman #43) Into a large dry test tube. 
Keep the funnel covered with a watch-glass to avoid loss by evaporation. 

Free Cholesterol Isolation 

1. Pipet 7.00 mi. of the filtrate into a heavy-wall conical centrifuge tube. Add 
3.5 ml. of digitonin solution and 2 drops of acetic acid 10% and stir well with a thin 
glass rod. Leave the rod in the tube and allow to stand in a closed Jar overnight, at 
room temperature. 

2. In the morning, transfer the tube to a rack, stir the contents gently to free pre- 
cipitate adhering to the wall near the surface of the liquid. Remove the rod and "file" It. 

3. Centrifuge the tube at high speed (2500 rpm) for 10-15 minutes to pack the pre- 
cipitate so that the supernatant can be decanted without loss of solid material. 

4. Decant the supernatant with a slow steady motion watching closely to ensure that 
no loss of solid material occurs. If any loss occurs the sample must be discarded and 
the determination repeated. 

5. Drain the tube in a vertical position for a few moments, remove the last drop by 
a clean cloth or paper. Return the rod to the tube, wash down the wall of the tube and 
the rod with 5 nil. of alcohol-acetone mixture and stir the precipitate thoroughly. Re- 
move and "file" the rod and centrifuge the tube for 5 minutes. Decant the supernatant. 

155 



6. Wash twice more In the same manner with ether. Return the rod to the tube. 
The tube and its contents may bo stored for several dayB at this stage. If color develop- 
ment la to be carried out immediately, the ether should be removed from the pre- 
cipitate by placing the tube in a moderately warm water bath for a few minutes. 

Precipitation of Total Cholesterol 

1. PIpet 3.00 ml. of filtrate Into a heavy -wall conical centrifuge tube. Add 3 drops 
of 33% KOH and stir with a thin glass rod until all droplets of the alkali have disappeared 
especially at the tip of the tube. 

2. Incubate at 38°C . for 30 minutes. Add alcohol -acetone to the 6 ml. mark, add 

1 drop of phenolphthaleln and titrate with acetic acid 10% until the pink color disappears. 
Add one more drop of acetic acid and 3.0 ml. of dlgitonln solution, Btlr well with the 
glass rod and allow to stand overnight as above under free cholesterol. 

3. The sample is washed as above under free cholesterol except that the precipitate 
is washed with ether only once. 

Development and Reading of the Color 

1. Add 2. 00 ml. glacial acetic acid and dissolve by the aid of heat (boiling water 
bath) and stir with the glass rod. 

2. Prepare similar tubes for standard and blank by pipetting 2. 00 ml. of glacial 
acetic acid (blank) and 2.00 ml. of cholesterol working standard (standard) into conical 
centrifuge tubes (heavy walled). 

3. Transfer the tubes to a water bath at 25°C. out of contact with direct sunlight. 
When the tubes are at 25°C. add to each in succession 4. 00 ml. of the acetic anhy- 
dride -sulfuric acid mixture, mix well with the glass rod and stand in the 25°C. water 
bath for exactly 30 minutes. 

4. Adjust the photometer to 100% transmittance at a wavelength of 625 mu with the 
blank tube and read each tube In order 30-31 minutes after the addition of the color 
reagent. The same photometer tube may be used for each reading without rinsing If 
the tube Is well drained between readings. 

Calculation : 

A. Free Cholesterol 

(D U /DJ x 0.2 x 100/(7/25) = mg. free cholesterol per 100 ml. serum. 

(D../D ) x 71.4 - mg. free cholesterol per 100 ml. serum, 

us - 

B. Total Cholesterol 

(D /D ) x 0.2 x 100/(3/25) - mg. total cholesterol ner 100 ml. serum 
(D /D ) x 166.7 s mg. total cholesterol/LOO ml. serum 

156 









Note : 

If the amount of serum is limited, the following modification may be used: About 
2 ml. of acetone-ethanol are placed In a graduated conical heavy-walled centrifuge tube 
and 0. 2 ml. of serum la added slowly In such a way that It runs down the wall of the 
flask and forms a layer under the solvent. The contents are Immediately mixed thor- 
oughly with a swirling motion. The solvent is brought just to the boil on a steam bath, 
and the tube is then cooled, and acetone-ethanol added to the 5.00 ml. mark. The 
suspension la thoroughly mixed and filtered into a small test tube through a fat-free 
filter. Two ml. of the filtrate are taken for free cholesterol and 1 ml. is taken for 
total cholesterol with the other reagents reduced approximately in proportion but with 
the technique remaining the same. Color development and reading are carried out as 
described above. 

Allowable variations in the procedure (Reference 4J: 

1. The proportion of Berum volume to extract volume may be decreased to compen- 
sate for exceptionally high cholesterol concentrations, but not increased. 

2. The volume of extract in which precipitation is carried out may be varied at will, 
provided that 1 drop of alkali solution per 1 ml. of the extract is added In the determina- 
tion of total cholesterol, and that 1 ml. of digitonin solution Is added for each 2 ml. of 
acetone-ethanol . 

3. The volume in which color is developed may be varied without reference to the 
volume in which precipitation waB carried out, provided that 2 ml. of the acetic anhy- 
dride-sulfuric acid reagent are added for each 1 ml. of acetic acid. 

A simple rapid procedure for the determination of total cholesterol is given by 
Carr and Drekter in Reference 5. It however, offers little or no advantage over the 
method of Schoenheimer and Sperry for the determination of free cholesterol. 

Purification of Petroleum Ether - Procedure : 

1. Petroleum ether must stand over H^SC^ for at least two weeks. 

2. Pour petroleum ether from bottle (being extremely careful not to get any 
acid over) and neutralize using 10% NaOH. Check pH with red and blue litmus 
paper. Discard acid. 

3. Siphon off the layer of NaOH (bottom). 

4. Wash petroleum ether 3 times with distilled water, siphoning off H 2 each time. 

5. Dry with approximately 200 g. Na 2 C0 3 . 

6. Filter through 12 inch filter paper Into flask for redistillation. 

7. Connect apparatus and redistill, ut.ing hot plate not flame. 

8. Discard first 50-100 ml. 

9. Check temperature during distillation. 30 60°C. 

157 



CONGO RED TEST 
Serum 

Reference: 

Paunz, I*., Magyar Orvosl Arch. 25, 499 (1924). Chem. Abstr. 19, 1009 (1925) 

P rinciple : 

In amyloidosis (deposition In various tltioues of a protein giving a reddish Iodine teat) 
congo red rapidly disappears from the Mood. la this condition 60-100 % will disappear In 
one hour; normally 15-30% disappears. Amyloidosis: occurs inpatients suffering from a 
long-standing suppurative (pus -forming) process and also ldiopathically. 

Apparatus : 

Colorimeter or Photometer. 

Reagents : 

1. 1% Congo red in water (sterile for injection) 

2. Acetone 

Procedure : 

1. Inject Intravenously for each 50 kg. of body weight, 6 ml. of 1. 0% congo red. 
Four minutes later collect 10 ml. of blood; 1 hr, after injection collect 10 ml. of blood, 

2. Allow blood to clot, centrifuge, remove the serum. 

3. To a measured volume of serum add 2 volumes of acetone. Shake well and centri- 
fuge while stoppered. 

4. Remove supernatant fluids and compare in colorimeter, or measure optical den- 
sity against a blank of 67% acetone. 

Calculation : 

A. Colorlmetrlc 

Depth of 4 m inute specimen x 100 _ % dye remaining in the plasma 
Depth of 1 hour specimen 

100 - pti- cent in plasma - % absorbed 

B. Photometric: (515 mu) 

B 1 hoMr x 100 = % dye retained m plasma 
D 4 mln. 

Example: ^-Pl? x 100 = 2 5% retention 
0.204 

100 - 25 = 75% absorbed 
Interpretation : 

See principled The urine may contain large amounts of the dye in a lipoid nephrosis. 

158 



( 






CREATININE (and CREATINE) - Serum and Urine 

References : 

1. Taussky, H.H. , J. Biol. Chem. , 208, 853(1954) 

2. Clark, L.C., and Thompson, H. L. , Anal. Chem. 21, 1218 (1949) 

3. Lfrfken, F. , Scand. J. Clin, Lab. Invest. 8, 325 (1954) 

4. Van Pilsum, J. F. , and Bovis, M. , Clin. Chem. 3, 90 (195T) 

Princip le: 

Creatinine reacts with alkaline plcrate solution to give a red color - the so- 
called Jaffe* reaction. This reaction 1b not specific for creatinine and other substances 
In blood, especially In the red blood cell also give a red color. However, the total 
chromogen expressed as creatinine is commonly used for clinical purposes, and a 
simple determination for both creatinine and Its precursor creatine Is given In Method L 
Method II is more specific for creatinine and Is recommended for the determination of 
creatinine clearances. 

Method 1 (References 1 and 2j 

Reagents : 

1. Picric acid 0. 040 JJ. Weigh out S. 16 g. of high grade analytical reagent picric 
acid, dissolve with stirring in about 900 ml. distilled water and dilute to 1000 ml. 

2. Sodium hydroxide 0.75 N. Dilute 30 ml. of 2.S N NaOH up to 100 ml. with 
distilled water. 

3. Creatinine stock standard. Dissolve 1.0000 g. pure dry creatinine (not the 
zinc salt) in 0. 1 N HC1 and make up to 1000 ml. with the acid. This solution Is stable 
almost Indefinitely. 1.810 g. creatinine - ZnCl 2 may be used. 

4. Creatinine working standard. Dilute 1. 00 ml. of stock standard up to 100 ml. 
with water. Mix well. Each ml. contains 0.01 mg. creatinine. 

6. Sodium tungstate, &%. Dissolve fi.O g. NajWO^HgO in water and make up 
to 100 ml. 

6. Sulfuric acid 0.67 N. See p. 30. 

Procedure ! 
Urine : 

1.' Dilute 5. 00 ml. of urine to 500 ml. in a volumetric flask. Mix well. 

2. Pipet 6.00 ml. of the diluted urine into a test tube and add 2.00 ml. of 0.04 N 
picric acid and 2. 00 ml. of 0. 75 N NaOH. Mix well. 

3. Treat 6.00 ml. of distilled water and 6.00 ml. of standard creatinine (working) 
in the samp - r ay. 

4. Allow to stand for 15 minutes and read photometrically within the next 30 min- 
ute 8, using 515 mu. 



159 



Serum 

1. Dilute 2.00 ml. of plasma or serum with 2.00 ml. of distilled water, add 2.00 
ml. of 5% sodium tungstate and 2.00 ml. of 0.67 H sulfuric acid, adding the latter drop- 
wise with shaking. Allow to Btand for ten minutes, shake again and filter. 

2. Plpet 3.00 ml. of filtrate Into a test tube, add 3.00 ml. of distilled water. 
Then add 2. 00 ml. of picric acid 0, 04 N and 2. 00 ml. 0. 75 N NaOH. Mix well. Allow 
to stand 15 minuteB and compare photometrically with a blank consisting of 3. 00 ml. 
of distilled water and a standard consisting of 2. 00 ml. of creatinine standard carried 
through all steps as given above (including Step 1), using 515 mu, 



Calculation : 
Urine: 



(D u /D g ) x 0.06 x 100/0.06 - mg. creatinine/100 ml. urine 
(D U /D B ) x 1000 = mg. creatinine per liter of urine. 



Serum: 



(D u /D g ) x 0.02 x 100/2 t mg. creatinine /100 ml. serum 

(D^D B ) = mg. creatinine/100 ml. serum 

Method (References 5 and 4) 

Principle : 

A trichloroacetic acid protein-free filtrate of serum in treated with Lloyd's 
reagent (a form of hydrated aluminum silicate) which adsorbs the creatinine, but not 
other plcrate chromogens of serum. The creatinine Is then eluted and the color devel- 
oped by alkaline plcrate solution. 

Reagents : 

1. Trichloroacetic acid IN. Weigh out 16.3 g. reagent grade CCl„'COOH in a 
beaker, add 100 ml. distilled water and dissolve without heat. 

2. Lloyd's reagent - each new bottle should be tested for its ability to adsorb 
creatinine. 

3. 0.04 N picric acid. A saturated solution of picric acid is prepared by adding 
about 16 g. of picric acid to one liter of water In a 2 L. flaBk. When heated to about 
80°C. complete solution takes place. Cool to room temperature (some acid will cry- 
stallize out). Dilute about 690 ml. of this saturated solution to one liter and titrate 
with 0. 1 N NaOH using phenolphthaleln as an indicator. The strength 1b then adjusted 
to between 0.0395 N and 0. 0405 N. 

4. Sodium hydroxide 0.15 N- Dilute 6 ml. of 2.5 N NaOH (see solutions p. 28). 
up to 100 ml. with distilled water. Titrate with standard HCl solution and adjust to 
between 0. 148 N to . 162 N. 

6. Alkaline plcrate reagent. Just before use, mix 40.0 ml. of 0.04 N picric 
acid and 60. ml. of 0. 15 N NaOH. 



160 






0. Crcntlntne-Btock standard. DIhhoIvo 1.0000 g. pure dry creatinine in approx- 
imately 0. 1 N fICl and dilute to the mark In a 1 liter volumetric flask using the same 
acid. 1.610 g. creatinine -ZnCl 2 may be used, 

7, Creatinine -working standard. Dilute 2.00 ml. of stock standard to 100 ml. 
with water. Mix well. 3.5 ml. contains 0.07 mg. (70 ug.). 5 ug./ml. will be the 
concentration In the final colored solution. 



Procedure : 

A. Deprotelnlzntion. To 3.5 ml. serum add 3.5 ml. H„Oand7.0ml. IN 
CCl 3 'COQH. Stopper. Shake vigorously, and allow to stand 10 minutes, again shake 
vigorously, and centrifuge for 10 minutes or until the precipitate Is well packed. Treat 
3. 5 ml. of water and 3. 5 ml. of dilute working standard similarly at the same time. 

B. Adsorption. To a test tube containing 200 mg. Lloyd's reagent transfer 10 ml. 
of supernatant (by use of cotton -tipped plpet). Stopper, and gently mix the contents of 
the tube once each minute for 10 minutes. Centrifuge and decant the supernatant and 
allow to drain 3-4 minutes. Discard the supernatant. 

C. Elutlon and color development. Add 10.0 ml. of the alkaline picrate reagent, 
mix thoroughly by shaking, and gently mix once each 2 minutes for 10 minutes. Centri- 
fuge and transfer sufficient of the supernatant for photometric comparison to cuvets 
(using a plpet). After another 30 minutes compare photometrically against the water 
blank and the standard using a spectrophotometer set at 515 mu. 



Urine : 

Dilute 6.00 ml. urine to 500 ml. in a volumetric flask. Mix well. Follow proce- 
dure as given under serum above, steps A. , B, , and C. 



Calculation : 
Be rum: 



(D /D ) x 0.05 x 100/2.5 - mg. creatinine/ 100 ml. serum 
u s 

(D u /D ) x 2 z mg. creatinine/ 100 ml. serum 



Urine: 



(D u /OJ x 0.05 x 100/0. Of ft * tng. creatinine/ 100 ml. urine 
(D„/D R ) a 200 m mtf. creatinine/ 100 ml. urine 



1«1 



CREATINE 
Urine and Scrum 
Principle ; 

Creatine In the urine 1b converted to creatinine by heating hi the presence of acid. 
After heating the total creatinine Is determined as above. The difference between total 
creatinine and pre -formed creatinine gives the value for creatine. 

Reagents : 

Aa for the determination of creatinine (Method I). 

Procedure : 
Urine : 

1. Plpet 6.00 ml. of diluted (1:100) urine Into a teat tube calibrated at 10.00 ml. 

2. Add 1.00 ml. 0.04 N picric acid. 

3. Place the test tube in a boiling water bath for forty -five minute t. 

4. At the same time treat a blank consisting of 1. 00 ml. of distilled water and a 
standard consisting of 6.00 ml. of working standard la the same way. 

5. Cool to room temperature after forty-five minutes. Add another 1. 00 ml. 
picric acid to each tube and mix well. Then add 2. 00 ml. 0. 71 g NaOH, add distilled 
water to the 10 ml. mark, mix well. Allow to stand for If minutes and read within the 
next 30 minutes. 

Serum: 

Treat a mixture of 3.00 ml. of 1:4 acid filtrate (one Method I) and 8,00 ml. of 
distilled water aa outlined above. 

Calculation : 

As under Creatinine (Method I) 

Total creatinine - preformed creatinine z creatine (aa creatinine). 

Creatine (as creatinine) x 1. ISO a creatine 

For a somewhat more elaborate procedure for »»mfa««»g; non-specific chromo- 
gen Interference In both urine and serum see Tanaaky , H. H. , Cllaica Chimlca Acta, 
1, 210 (1966). 

Interpretation : 
Urine : 

Creatine phosphate in the muscle serves as a reservoir tor high energy phosphate. 
During muscle contraction creatine -phosphate is broken down to creatine and phosphate. 
Creatinine is derived from creatine at a very steady rate which is uninfluenced by most 
metabolic changes. Creatinine Is a waste product and is found in the' urine In amounts 
which are very constant from day to day for any given individual. The normal daily uri- 
nary excretion is between 1 and 2 grams, higher In men than In women. 

The normal creatine excretion Is very low or completely absent, except In children 
and In women in pregnancy and for a short time after delivery. Taussky (1) found from 
to 600 mg. creatine excreted per 24 hours. 

162 



Creatinine excretion changes very little with disease, except in very severe 
kidney dysfunction. 

Creatine excretion may Increase In muscular disorder such as myasthenia 
gravis, muscular dystrophics, etc. Any rapid decrease In the muscle mass may con- 
tribute to an elevated urinary creatine. 

Serum: 

By Method I. , serum creatinine values between 1 and 2 mg. per 100 ml, are 
found. By Method II. , rather leas, 0, 7 to 1.4 mg. /100 ml. are found. This Is a 
reflection of the fact that some of the Jaffe positive material in plasma Is not crea- 
tinine but substances giving the flame color. In the red blood cell, almost half of the 
chromogenic material is nou-creatlnine in nature. 

Most of the creatine is found in the cells; whole blood creatine being from 2 to 
7 mg. % normally; and plasma creatine Is well under 1 mg. %. 

Plasma creatinine Increases In renal disease, somewhat later than does urea. 
More than 5 mg.% in a chronic nephritis Indicates a poor prognosis. Acute renal 
syndromes may show much higher creatinine values and still have a good recovery. 
Plasma creatine determinations have been little used. There is some evidence that 
creatine levels are Increased In hyperthyroidism. 

Creatinine clearance studies have been widely used as a measure of glomerular 
filtration rate. In dogs inulln and creatinine clearance studies give identical results. 
In man, with normal plasma creatinine levels, this is approximately true, but with 
elevated plasma levels, the creatinine clearance does not represent glomerular fil- 
tration alone, since some tubular excretion la taking place. 









.. 



163 



ESTERASE (TRE3UTYRINASE) 












Serum 

References : 

1. Goldstein, N. P., Epstein, J, H. . and Roe, J. H. , 
J. Lab, & Clin. Med. 33, 1047 (1048). 

2. Alper, C. , Standard Methods of Clinical Chemistry _1, 71 (1963), 

3. See also references under Lipase - Serum 

Principle ; 

Esterase of serum Is allowed to catalyze the hydrolysis of the ester bond in trlbu- 
tyrin (glyceryl tributy rate) . The acid liberated is quantitatively estimated by titration 
with standard alkali. 

Reagents : 

1. Calcium acetate solution 2%. 

2. Sodium diethylbarbiturate solution 0.6%. 

3. Sodium choleate, solid. 

4. Tween 20. 

5. Methocel (15 centipolse) 

6. Tributyrin, c. p. This may be purified by neutralizing with sodium bicarbonate, 
washing with distilled water and distilling under reduced pressure. 

7. Esterase substrate. 

Add 250 ml. Z% (w/v) calcium acetate solution to 260 ml. 0.6% (w/v) sodium 
diethylbarbiturate solution, Add to this mixture, 6.7 mg. sodium choleate, while beat- 
ing in the Waring blender. Add 0.26 ml. Tween 20, and 0.6 g, Methocel, Continue 
beating and add 6. 25 ml. tributyrin drop wise from a buret. Mix by beating 3. 5 minutes. 
Adjust by NaOH to pH 8.56. Store In refrigerator. This emulsion should be good for 
at least 14 days. 

8. Ethanol-ether 9:1. Add 100 ml. of a diethyl ether to 900 ml, of 96% ethanol. 
Mix well. 

9. Alkali 0.05 N. Either sodium or potassium hydroxide may be used. 

Procedure : 

1. Measure 20 ml, portions of esterase substrate into each of two large test tubes. 
Warm the tubes to 37°C. in a water bath. 

2. Add 1.0 ml. of serum to one tube, mix well and incubate both tube* at 37°C. for 
1 hour. 

3. At the end of the 1-hour incubation period, add 1.0 ml. of serum to the control 
tube and Immediately pour the contents of each tube into a 100 ml. beaker, and trans- 
fer completely by rinsing each tube with 60 ml. of 9:1 alcohol ether mixture. Mix veil 
with a glass stirring rod. 

4. Titrate the mixture with 0. 05 g alkali to a distinct pink with phenolphthalein 
as the Indicator. If a pH meter is used, titrate to pH 10. 85. 



164 



I 






Calculation : 

See Lipase, p. 208, 

Interpretation : 

There are present In human sera a number of enzymes capable of splitting the 
ester bond which have been classified at* follows: 

1. Choline sterases 

a. True acetylcholinesterase 

This enzyme is found In large quantities In brain and In erythrocytes, ft If 
Inhibited by relatively low concentration of eserine and by relatively low concentration 
of substrate. 

b. Pseudo-cholinesterases 

These are found In tissue and In serum. They are characterized by the high 
concentration of eserine and of choline-eater substrate required for inhibition. The 
esterase measured by the procedure described above falls into this class, 

2. Ali-este rases (simple esterase) 

These are enzymes catalyzing the hydrolysis of esters of low molecular weight 
fatty acids and differing from pseudo-cholinesterases in that they are not Inhibited by 
eserine. 

3. Lipases 

These are enzymes catalyzing the hydrolysis of esters of high molecular weight 
fatty acids - such as olive oil, etc. 

4. Sterol esterases 

These are enzymes catalyzing the hydrolysis of (e.g.) cholesterol esters. 

The variations with disease of these various types of esterases have not been well 
studied. The following gives a general outline of the data obtained. 

1. Pancreatic disease - In acute exacerbations of chronic relapsing pancreatitis, 
In acute pancreatitis, and in obstructive pancreatic duct lesions, the li pase is elevated, 
the e sterase is normal, 

2. Liver disease - In some types of toxic liver disease there may be decreased 
serum amylase and lipase but increased pseudochoUnesterase. In moderate and severe 
cirrhosis, the levels are below normal. 









166 



EBTROQENS 
Urine 

References : 

Anker, Rudolph M. : Determination of estrogens In stored urines of pregnancy, 
J. Clin. Endo. & Me tab. 15, 210-214 (1955). 

Bachman, Carl, Pettlt, Dorothy 8. : Photometric determination of estrogens, 
m. A procedure for the estimation of the estrogen* of pregnancy urine, J. Biol. 
Chem. 138, 689-704 (1941), 

Principle : 

A 100 ml. aliquot of a 24-hour urine specimen is taken for assay. The determina- 
tion comprises (a) hydrolysis of the water-soluble conjugates in urine; (b) extraction 
of the steroid moieties and their separation by their partition between solvents} and (c) 
spectrophotometry estimation of the color developed by sulfuric acld-ethanol reagent. 

Reagents : 

1. Ethyl ether: absolute, peroxide -free. 

2. Hydrochloric acid: concentrated, reagent grade. 

3. 9% sodium bicarbonate: 9 g. reagent grade NaHCO„ in 91 ml. of distilled 
water. 

4. 9% sodium carbonate: 9 g, of reagent grade, anhydrous, sodium carbonate In 
91 ml. of distilled water. 

6. Benzene: reagent grade. 

6. IN sodium hydroxide: 20 g. of the reagent grade pellets of NaOH are dis- 
solved in 600 ml. of distilled water. 

7. 45% sulfuric acid: 90 ml. concentrated, reagent grade acid is carefully added 
to 110 ml. of distilled water and cooled to room temperature. 

8. Ethanol: absolute, dehydrated, reagent of NF grade. 

9. Color reagent A: Carefully and slowly add 80 ml. of concentrated sulfuric acid 
to 20 ml. absolute ethanol. Cool to room temperature and store In refrigerator. This 
reagent Is good for 14 days. 

10. Color reagent B: carefully add 00 ml. of concentrated sulfuric acid to 30 ml. of 
absolute ethanol. Cool to room temperature and store In refrigerator. This reagent is 
good for 14 days. 

11. Standard estradiol: 10 mg, of pure estradiol In 100 ml. of 95% ethanol. 

Procedure : 

1. The total volume of the specimen (preserved with 15 ml. of toluene) Is recorded. 
Carefully measure out a 100 -ml. aliquot of the specimen using a graduated cylinder. 

2. Place the aliquot In a 250 ml. Erlenmeyer flask with two glass beads to prevent 
bumping, add 15 ml. hydrochloric acid. Bring the contents of the flask to a boll, and 
continue to boil GENTLY for 10 minutes. When boiling is completed, cover neck of flask 
with a 50 ml. beaker, ami chill contents of flask to below 16° C. In the refrigerator. 

3. Extract the hydrolyzed urine in a 250 ml. separatory funnel with one 100 ml. , 
and two 50 ml. portions of ether. Combine ether extracts; discard extracted urine. 

166 






< 



4. WaHh the ether extract once with 8 ml. of sodium bicarbonate, In the separatory 
funnel. Allow to nettle five minutes. Discard uqueoitB phase. 

5. Evnporttte the ether extract to drynesB In a 250 ml. glass evaporating dish In a 
hood. Avoid flamcR. Dissolve residue In 0. 5 ml. of ethanol and add 35 ml. of benzene. 

6. Extract the benzene solution with one 35 ml. and two 17 ml. portions of 9% sod- 
ium carbonate and one 5ml. portion of water. The benzene contains the hormones es- 
trone and estradiol. The aqueous phase contains the hormone, estrlol. 

Estrlol Phase 

1. Acidify the aqueous phase from the above step to litmus paper using hydrochloric 
acid. 

2. Extract the acidified solution with three 40 ml. portions of ether. Discard aqueous 
phase; combine ether extracts. 

3. Wash ether extract with 12 ml. sodium bicarbonate; discard the aqueous phase. 
Evaporate ether extract to dryness. 

4. Dissolve residue in 0.5 ml. ethanol, dilute with 35 ml. of benzene. Wash solu- 
tion once with 1 ml, of sodium bicarbonate and discard washing, 

5. The estrlol Is transferred to water by washing the benzene with three 50 ml. 
portions of water. The combined water extracts are evaporated to dryness and the resi- 
due taken up in 2 ml. of ethanol for final assay. 

Estrone-Estradiol Phase 

1. The benzene solution from Step 6 is washed with 10 ml. of 46% sulfuric acid fol- 
lowed by two 20 ml. portions of water. The washings are discarded. 

2. The estrogens are extracted from the benzene with four 35 ml. portions of 1 N 
sodium hydroxide. The benzene is now discarded. Make the alkaline extract acid to 
litmus paper using hydrochloric acid. 

3. Extract the acidified sodium hydroxide with one 100 ml. and two 50 ml. portions 
of ether. Combine the ether extracts; discard the aqueous phase, 

4. Wash the ether extract with 15 ml. of 45% sulfuric acid. Next wash with two 25 
ml. portions of 9% sodiur" carbonate, followed by two final washings with 25 ml. of 
water. Discard all washings. Evaporate ether extract to dryness In a 250 ml. glass 
evaporating dish. 

5. The residue is taken up in two ml. of ethanol, and stored in a stoppered cylinder 
until final assay. 

Color Development 

1. The alcoholic urine extracts from both the Estrlol and Estradiol-Estrone frac- 
tions are placed in test tubes appropriately marked. The full 2.0 ml. volume of 
alcoholic urine extract Is used In each case. 

2. 0. 25 ml. of alcoholic estradiol standard is placed in a third teBt tube and ap- 
propriately marked. 

3. Evaporate the contents of all tubes to dryness in either a vacuum desiccator or 
a gentle stream of air. 

4. To each tube add 2.0 ml. of Color Reagent A. Stopper each tube loosely to 



167 



exclude moisture. Heat In a boiling water bath for 10 mtnutca, shaking well at two 
minute intervn la. The color, which In quite stable, will develop 'at this point. Cool 
nil tubes to room temperature. 

6. Quantitatively transfer the contents of each tube to 10 ml, volumetric flasks. 
DUute to the mark with color reagent B, Mix thoroughly. 

6. The optical density Is determined at 406 mu. , 466 mu. , and 505 mu. on the 
Reckman Spectrophotometer using 1 cm. quartz cells against 3 ml. of color reagent li 
as the blank. 



( 



Calculations ; 

To calculate the amount of estrogen present, a correction for extraneous non- 
specific color must first be made using the following equation: 



(1) 



CD. 



(456) 



= OD 



cm) 



^ OD (408) * OD (506)N 

v a J 



The corrected optical density at 466 mu. (CD^gg) Is substituted in the following equa- 
tion to find the amount of Estrogen present in the 100 ml. aliquot of urine used. 



(2) 



C p 456 
E, 



micrograms % (tig %) 



The factor Ef In the above equation varies with the fraction under determination, and 
represents the optical density for 1 microgram of the estrogen diluted to 10 ml. as Is 
done In the outlined orocedure. 

E f 

Estrlol Fraction - 0.013 

Estrone -Estradiol Fraction - 0.018 

Standard Estradiol - 0.009 






( 



In calculating the 25 ug standard the results should not vary more than from 24-26 
micrograms. If a greater or lesser result is obtained, Color Reagents A and B prob- 
ably have become too decomposed, and fresh solutions should be prepared with the 
procedure repeated over from the beginning. 

Lastly, the total amount of Estrogen In the 24-hour specimen is calculated: 



(3) 



J!S% 



100 



x total volume ■ total Estrogen (In micrograms) 



The results are reported on Standard Form 514a in the following manner: 






Total volume 
Estrlol 



Estrone-Estradiol 
Total Estrogen 



ml. 

micrograms 

micrograms (calculated as estrone) 

micrograms 



168 






Example; A 24 -hour specimen of urine of total volume 1000 ml. gave the following 
readings in optical density on the Beckman Spectrophotometer. 



Fraction 
Estrone -Estradiol 
Estrlol 
Standard Estradiol 



466 mu 
0.615 
0.567 
0.305 



The results were calculated In the following manner: 
Estrlol Fraction 

(1) CD 456 x 0.B67 - 0.873 { 0.0™ 



406 mu 

0.801 

0.973 

0.125 



506 mu 
0.169 
0.073 
0.061 






CD 456 s 0.567 - 0.623 - 0.044 

(2) 0.044 _ 3<4 
0.013 



(3) 3.4 
100 



x 1000 s 34 micrograms 
Estrone -Estradiol Fraction: 



U) CD 4gfl = 0.615 - 0.801 / 0.160 
CD 466 a 0-816 - «- 4 85 m. 0.130 

(2) 0.130 « 7.2 
0.018 

(3) JJL x iooo - 72 micrograms 

100 

Standard Estradiol : 

it% nn n ans 0.125 £ 0.051 
(1) CD 456 = 0.305 - £ 



(2) 0.217 
0.009 

Results: 



- 0.305 - 0.088 - 0.217 



24. 1 micrograms 



Total volume 



1000 






169 






Estrlol 


34 


Estrone-Estradiol 
Total Estrogen 


74 
108 



ml. 

micrograms 

micrograms (cal. as estrone) 

micrograms 



Notes : 

This procedure Is primarily designed for estrogen as«ay of urines of late preg- 
nancy or urines In which a high amount of estrogen excretion is suspected. Normal 
male and female urines usually fall to give results above zero because of excessive 
chromogenlc interference. In calculating the results, equation 91 will usually give a 
negative value In these cases which does not necessarily indicate the absence of estro- 
gen. Satisfactory results will usually be obtained, however, when the total 24-hour 
estrogen excretion is 100 micrograms or higher. 

Id reporting the results of assay of normal urines, when Equation #1 gives a 
negative value, the results of analysis may be reported as "Total Est^o^en content 
does not appear to exceed the normal limits. " 



Interpretation: 

Normal Values: 






Total Estrogen In 
Female micrograms per day 

Normal Adult before Menopause - 20 to 80 

Normal Adult after Menopause - below 50 

Pregnancy - *see note 






Male 

Normal Adult - to 6 



< 

♦During pregnancy the total estrogen rises to about 12 to 40 milligrams 
per day, with estrlol comprising about 00% of the total amount. 
Estriol Is usually absent In normal female urines. 












170 















FAT IN FECES 

Reference b : 

Hepler, O. E. Manual of Clinical Laboratory Methods, Springfield, HI. Charles 
C. Thomas. 4th Ed. (1949). 

Fowweather, F. S. and W. N. Anderson, A method for the determination of fat 
In feces. Blochem. J. 40, 350-351 (1946). 

Van de Karver, J. H. , Halnlnk, H. Ten and Weyers, H. A. , J. Blol Chem. 177 . 
347 (1949) . 

Principle : 

Total fat includes free fatty acids, soaps, and neutral fat fractions. Hydrochloric 
acid Is added to a portion of feces to convert the soaps to fatty acids. The total fat Is 
then removed In an ether extraction, purified by means of petroleum ether, and deter- 
mined gravlmetrlcally. Another portion of feces (not treated with hydrochloric acid) Is 
extracted with ether and the amount of free fatty acids and neutral fats are determined 
gravlmetrlcally. From this precipitate the free fatty acids are dissolved In benzene 
and titrated with 0. 1 N sodium alcoholate. 

Reagents : 

1. Concentrated hydrochloric acid. 

2. Anhydrous ethyl ether, 

3. 96 per cent ethyl alcohol. 

4. Petroleum ether - boiling point should be below 60° C. 

5. Benzene. 

6. 0.1N sodium alcoholate 

a. Place about 500 ml. absolute ethyl alcohol (redistilled) In a 1 liter volumetric 
flask. 

b. Add 2.3 grams freshly cut metallic sodium; when dissolved, dilute to volume 
with alcohol. Keep away from flames. 

c. Titrate with 0. 1 N hydrochloric acid using 2 drops 0.5 per cent alcoholic 
phenolphthalein as indicator. 

d. Adjust normality to 0. 1 with alcohol as the solvent. 

Procedure : 

1. Record total weight of fresh specimen. 

2. Thoroughly mix and homogenize specimen to afford even distribution of contents. 

3. Weigh onto two previously tared aluminum dishes about 2-3 grams feces from 
specimen. Label 1 and 2. 

4. Place dishes on hot plate In hood until all odor Is dispelled. Then continue heating 
in drying oven (95 to 100° C.) until samples are at constant weight. From final weighing 
calculate DRY WEIGHT and MOISTURE CONTENT of specimen. 

5. Weigh two samples of about 2-3 grams fresh specimen Into two 250 ml. centrifuge 
bottle b. Label A and B. 

6. To bottle A add 3 ml. concentrated hydrochloric acid and 30 ml. distilled water. 
Bottle A Is used for determination of TOTAL FAT. 

171 



7. To bottle B add 30 ml. distilled water. Bottle B 1b used for the determination of 
FATTY ACDDS and NEUTRAL FAT. 

8. Add 20 ml. anlwdroua ethyl ether to each bottle and shake for five minutes. (Use 
caution to avoid loss of ether.) Cool under tap if nficessary. Let stand 6 minutes. 

9. Add IT ml. 95 per cent ethyl alcohol to bottle A and 20 ml. to bottle B. Mix con- 
tents by a quick rotary motion and cool to room temperature In running water. 

10. Stopper bottles and shake vigorously for 5 minutes. Then centrifuge at 2000 rpm. 

11. Transfer the ether layer, as completely as possible, into 150 ml, beakers, 
(Label A and B). Repeat extraction twice using 20 ml. portions of ether. Combine the 
three extractions. 

12. Wash the stopper and sides of the bottles with three successive 6 ml, portions of 
ether and add each to the ether extractions. 

13. Evaporate the combined extractions and washings to dryness. 

14. Add 20 ml. petroleum ether to each beaker, warm on a water bath, and filter 
through fat-free filter paper into two previously tared 100 ml. beakers. Repeat twice 
using 10 ml. portions of petroleum ether. Label A and B. 

15. Evaporate the petroleum ether to dryness. Dry residue to constant weight In a 
37° C. oven. Record weights. 

16. Dissolve residue in beaker B in 50 ml. benzene and heat almost to boiling. 

17. Titrate while still hot with 0.1N sodium alcoholate, using two drops alcoholic 
phenolphthaleln as Indicator. Titrate until color no longer deepens. 

Calculations : 

1. Dry Matter: dried weight sample 1 x 100 _ peT cent ^ ma tter 

wet weight sample 1 

(Repeat with sample 2 and take the average) 

2. Total Fat: 
a. Calculate the weight of dry matter In Barapie A by multiplying the wet weight 

by the per cent of dry matter found. 

b total fat "(sam ple A) x i 00 = per cent total fat 
dry weight (sample A) 

3. Free fatty acids plus neutral fat: 
a. Calculate the weight of dry matter In sample B by multiplying the wet weight 

by the per cent of dry matter found. 

b free fatty acids plus neutral fat (sample B) x 100 equala per cent free 
dry weight (sample B) 

fatty acids and neutral fat. 

4. Soaps: 
a. (Per cent total fat) minus (per cent free fatty acids plus neutral fat) equals 

per cent soaps. 

172 



5. Free falty acids: 

a. 1 ml. of 0, 1 IJ sodium alcohol ate titrates 28, 2- mgm. of oleic acid or 28.4 
mgm, of stearic acid (average 28.3). 

b. The number of mis. of 0. 1 N sodium alcoholate used in titration times 28.3 
equals mgm. of free fatty acids In sample 1). 

c. Convert to grains and 

weight of free fatty acids (sample B) „ - ftft .- .... .. 

6 — - — -f- t - - v . ~. . g ' x ioo per cent j ree fatty acJdg 

dry weight (sample B) v 

8. Neutral fat: 

a. (Per cent free fatty acids plus neutral fat) minus (per centfree fatty acids) 
equals per cent neutral fat. 

7. Consolidate report as follows: 

Total weight of specimen ....... grams 

Moisture , per cent 

Dry weight of specimen grams 

FAT CONTENT (based on dry weight) 
Soaps (calculated as Oleic 

and Stearic acid) per cent 

Free fatty acids , per cent 

Total fatty acids per cent 

Neutral fat per cent 

Total fat per cent 

Notes : 

1. The analysis should be made on fresh feces an fat decrease! cm standing even 
when specimen 1b frozen. 

2. Blank determinations should be run to rule out any fatty substances in the reagents. 

3. The amount of dry matter, and likewise the moisture content, la extremely vari- 
able In the normal stool. Values should be expressed in terms of dry matter to be signi- 
ficant. 

4. Drying alters the chemical composition of feces so the lipids are determined in 
"wet" feces but reported as pfif cent of "dry" weight of feces. 

Interpretation: 

1. Normal values: 

a. Dry matter: 4.6-38 per cent 
Total fat: 7. 3 - 27. 6 per cent 
Free fatty acids: 1. 05 - 10 per cent 
Soaps as fatty acids: 0.64 - 11.4 per cent 
Neutral: 2. 49 - 11. 8 per cent 

Neutral fat as per cent of total fat: 24. 5 - 80. 1 per cent 
2. Abnormal values 

a. Celiac disease and obstructive Jaundice: increase in total fat, soaps and 



173 



free fatty acids; neutral fat is normal. 

b. Pancreatic deficiency: Increase In total fat, neutral fat and soaps with normal 
or low fatty acids, 

c. Nontropical sprue: increase in total fat, fatly acids and neutral fat. 

d. Tropical sprue: increase in total fat and fatty acids, and decrease in neutral 
fat. 

e. Gastroenteritis: increase in all fat fractions. 










































174 



C 



( 






FIBRINOGEN 



Plasma 
(Turbtdlmetric) 

Reference i 

FowelL, A. H. , American J. Clinical Pathology, 25, 340 (1B55) 
Parfentjev, LA., et al. , Arch. Biochem. 46, 470 (1963) 

Principle : 

Fibrinogen Is a globulin more easily precipitated than the other plasma globulins. 
It Is practically quantitatively precipitated by 12% (NH 4 ) 2 SO.. The plasma Is mixed 
with an appropriate salt solution and the resulting turbidity Is read in a photometer 
against the same plasma diluted in 0.85% saline. The fibrinogen is calculated by means 
of an equation determined empirically using purified fibrinogen diluted with fibrinogen 
free serum. Since fibrinogen does have some solubility in the reagent, a simple factor 
cannot be used, — see Notes below. 

Reagents : 

1. 0.85% NaCl; Dissolve 8,50 g. NaCl (reagent grade) up to one liter with distilled 
water. 

2. Precipitant solution: To 133.33 g. reagent grade ammonium sulfate and 10.0 g. 
NaCl, add 0.025 g. Merthlolate to suppress mlcrobical growth. The volume is brought 
to one liter with distilled water and sufficient 10 N NaOH added to bring the pH to 7 (use- 
Ing 0. 02% phenol red as an outside indicator), 

3. Serum (for standardization): Pooled normal human serum (from well coagulated 
blood specimens). 

4. Fibrinogen (purified) with clottable protein assay. Warner Chilcott brand was 
used to calibrate this procedure. 

Procedure : 

Plasma for this determination Is collected preferably in sodium citrate but oxalate 
or EDTA (Ethylenedlamlnetetraacetlc acid), dl-Bodlum salt, may be used, To 0.50 ml. 
of plasma (carefully separated and free of erythrocytes), In a photometer tube (22 x 
200 mm.), is added 6.00 ml. of precipitant solution. For a blank add to 0.60 ml. of 
the same plasma 6.00 ml. of 0. 85% saline. After three minutes compare photometrical- 
ly setting the blank at 100 using (515 mu). 

Calculation: 



Read the percentage fibrinogen from a graph derived from the standardization pro- 
cedure given below. Note that the line does not pass through 0,0. 

Standardization 

1. Dissolve gently a vial of fibrinogen containing 6 mg. clottable protein (fibrino- 
gen) In exactly 2.00 ml. of fibrinogen free serum. 

2. Into three photometer cuvets add as follows, using serum and the above recon- 
stituted plasma (serum and fibrinogen): 

177 



Tube Fibrinogen 



ml. 
Serum 



Reconst. ' Plasma Precipitant Solution 



( 



1. 


0.00 


1.00 


0.00 


2. 


0.07S 


0.75 


0.25 


3. 


0.150 


0.50 


0.50 


4. 


0.30 


0.00 


1.00 






12.00 
12.00 
12.00 
12.00 



Mix each tube Immediately after the addition of the precipitant and after exactly 
three minutes, mix and compare photometrically using 515 mu. 

Notes : 

1. Fibrinogen levels lower than a certain value (approximately 0.08%) cannot be 
measured by this method. 

2. If flocculation has occurred at the time of reading give the sample a vigorous 
shake to re suspend the fibrinogen. 

Interpretation: 

Normal levels of fibrinogen range from 0. 113-0. 380% averaging 0. 246%. 

Elevated values are seen In most acute Infections and may be correlated with 
Increased sedimentation rates. 

Decreases may be seen In very severe hepatic deficiencies or in congenital 
defects in synthetic metabolism , intravascular clotting, generalized, or in localized 
large scale fibrin formation. 
























178 



FOLLICLE STIMULATING HORMONE - Btoassay 

Urine 

Referen ces: 

Dekanskl, J, , The kaolin -adsorption method for the quantitative assay of urinary 
gonadotropics. Brit. J. Exp. Path. 30, 272-82 (194B). 

Crooke, A.C., W.R. Butt, J.D. Ingram, and L.E, Romanchuck, Chemical assay 
of gonadotropin In urine. The Lancet. JL, 379-83 (1954). 
Principle : 

Since there is no specific chemical reaction by which gonodotrophlnB can be mea- 
sured, the process described is essentially an extractive process using kaolin as an 
adsorbant of the gonodotrophlns and subsequent re -precipitation of the extract. An eluted 
extract is then assayed by its activity in mice. 

Apparatus : 

For each determination, four Immature female white mice are needed. 

Reagents : 

1. Glacial acetic acid 

2. Hydrlon or alkali-acid paper 

3. Sodium chloride 

4. Ethyl alcohol 95% 

5. Kaolin 20% (w/v) aqueous suspension 

6. Ammonium hydroxide IN. (67.5 ml. diluted to 1 liter) 

Procedure : 

1. The total volume of the urine specimen is poured into a large graduated cylinder 
and measured. If more than one specimen is run at one time, the volumes are made 
equal by an addition of distilled water. 

2. Adjust the pH of the urine to 4.5 with glacial acetic acid. Use either pHydrlon 
or alk-aeld paper. 

3. Add 5 ml. of 20% Kaolin suspension for each 100 ml. of specimen. (Equalizing 
the volumes allows equal amounts of kaolin to be added to each specimen). Add 1-2 

g. of sodium chloride and allow to settle in the refrigerator for about one hour. 

4. Siphon off the supernatant liquid being careful not to disturb the settled kaolin. 

5. Transfer the kaolin to a 250 ml. centrifuge bottle with distilled water and centri- 
fuge at 1500 rpm for about 10 minutes. Decant and discard the supernatant liquid. 

6. IN NH4OH is added to the residue In the bottles in a volume which equals the 
volume of the original kaolin suspension added. The precipitate is stirred well into 
the NH4OH with a stirring rod which Is removed and saved, 

7. Centrifuge and decant the supernatant Into a beaker. 

8. Repeat step #6. and combine the supernatants, 

9. Centrifuge the combined supernatants. Discard the precipitate and adjust the pH 
of the liquid to 8. 5 with glacial acetic acid using Indicator paper. 

10. Centrifuge and discard the precipitate formed at pH 8.6. Reduce pH to 5.5 with 



179 



acetic acid. Add a volume of 95% ethyl alcohol (or acetone) which Is equal to four time 8 
* ■» volume of the acidified solution, 

11. Place in a glass stoppered Erlcnmeyer flask and allow to stand overnight in the 
refrigerator. 

12. Centrifuge the entire sample using the some centrifuge bottle over and over 
discarding the supernatant after each centrifugatlon. 

13. Place the bottle In a vacuum dessicator and evacuate until dry. 

14. When the protein in the bottle is dry (usually overnight) add 7,5 ml. of distilled 
water. Stir well by swirling and then centrifuge. Take off the supernatant with a drop- 
per plpet and dilute It as follows: 

(1) 4 ml. extract / 1 ml. of H„0 - 3 Mouse Units Level 

(2) 1.67 ml. extract / 3. 33 ml. H„0 -9 " « " 

(3) 0.58 ml. extract /4.42 ml. rijjO -27 " " " 

(4) 0.19 ml. extract / 4; 81 ml. HJO -81 " " " 

15. Inject each twice daily with 0.5 ml. of each dilution for two days. Inject mice 
once on third day and sacrifice on the fourth day. The weakest dilution at which the 
uterus and Fallopian tube are twice the normal size gives the titer of the extract. 

Notes : 

The urine sample must consist of a 24-hour specimen and if shipped from a distance 
greater than 24 hours traveling time, 15 ml. of chloroform may be added as a preserva- 
tive. 

Interpretation: 

The normal male and female give negative tests except as Indicated. 
Tests may be positive in 
Males with 

Testicular hypofunction 

Seminoma or teratoma of testes 
Females in 

Pregnancy 

Menopause 

Ovarian hypofunction 

Cho r ionepithel i oma 

Hydatidiform mole 

and other disease , primary and secondary of the pituitary gland. 



180 



( 



( 



GLUCOSE 
BUxid and Spinal Fluid 

Reference : 

Nelson, Norton: J. Biol. Chem. , 1J53, 375-3S0 fl?M) modified and adapted. 

Principle: 

— ... ... ■ » . . . . 

The glucose in a blood filtrate is oxidized by cuprlc Ion (Cu^r) in an alkaline me- 
dium, and the cuprous oxide produced is then reacted with acid arsenoniolybdate to 
produce a solution of blue molybdenum oxides whose concentration Is directly propor- 
tional to the amount of glucose present In the sample. Blood fot glucose determinations 
should be collected in fluoride tubes to avoid the loss of glucose by bacterial and ery- 
throcyte glycolysis. 

Reagents : 

1. Deproteinizing solutions: see under PROTEIN-FREE FILTRATES for the prin- 
ciples involved and solutions required. 

2. Alkaline Copper reagent - for rolorlmetric technique. 

28 g. Na 2 HP0 4 anhydrous 
100 ml, 1 N NaOH or 40 ml. 2. 5 N NaOH 
40 g. KNaC 4 H.O -4H 2 {sodium potassium tartrate) 
8 g. CuS0 4 -5H 2 <J 
180 g. Na 2 S0 4 anhydrous 

Dissolve phosphate and tartrate in 700 ml. of water; add the NaOH; then add 
80 ml. of 10% CuS0 4 ; then add the sodium sulfate, stir until dissolved. 
Dilute to 1 L. Let stand 1-2 days, decant and filter. 

3. Arsenomolybdate color reagent: 

Dissolve 25 g. of (NrL^g MOijOo 4 '4 HLO (ammonium paramolybdate) In 450 ml. 
of water. Add 21 ml, of concentrated H 2 SO . and mix. Dissolve 3 g. of Na 2 HAsO.* 7H 2° 
In 25 mi. of water and mix the two solutions. Place In Incubator at 37°C. for 24-48 
hours. 

4. Glucose stock standard: 

Dissolve exactly 1. 000 g. of pure anhydrous glucose In 0. 25% benzoic acid, 
dilute with 0.25% benzoic acid to 100.0 ml. and mix. 

5. Glucose dilute working standard: 

Add exactly 1, 00 ml. of stock standard to a 100 ml. volumetric flask, add 
0, 25% benzole acid to the mark and mix well. This results In a 10 mg.% glucose 
solution, Prepare fresh each week, 

6. 0.25% benzoic acid solution: 

In a 2 liter volumetric flask, dissolve 5 . g. benzoic acid. Mix well. 

Procedure ; 

1. Pipet 1.00 ml. of a protein -free filtrate into a 26 ml. Folln-Wu sugar tube. 

2. Prepare a blank using 1.0 ml. distilled water and a standard by using 1.00 ml. 
of dilute (10 mg./lOO ml.) glucose standard. 



181 



3. To each of the three above In a 25.0 ml, Folln-Wu tube add 1.0 ml, mixed 
>pper reagent; mix by (ihakJnij, 

4. Cover with aluminum foil caps or glass marbles and Immerse simultaneously 
In a boiling water bath, for 10 minutes. 

5. Cool for three minutes In a pan of cold water. 

C. Add 1.0 ml. of aroono-rnolybdate reagent to each tube, mix and dissolve the 
cuprous oxide by staking. 

7. Let stand 10 minutes and dilute to the 25 ml, mark and mix by several Inver- 
sions. 

8. Read In the photometer (015 mu) Jusing the blank to set 100. 

Calculations : 

(D u /D g ) x C B x (100/0.1) s C u = (D u /D fl ) x 100 (mg./lOO ml, blood) 

Example: 

T u = 30.2; D u = 0.520 c fl = 0.1 mg. 

T a = 26.8 D„ = 0.672 

(0.520/0.672) x 0.1 x (100/0,1) - (0,520/0.572) x 100 * 91 mg.% 

Precautions: 

1. Be sure boiling water bath Is really boiling. 

2. Time boiling accurately. 

3. Mix well each time as directed. 

Interpretation: 

Normal blood sugars for Folln-Wu tungstlc acid filtrates are between 80 and 120 
mg. /10Q ml. ; for Zn(OH) 2 filtrates 60-90 mg.%. True fermentable blood sugar Is 
60-80 mg.%. Increases are seen after Ingestion of food, 'In diabetes and In emotion- 
al stress. Decreases are seen In Insulin overdosage and with active Islet cell adenom- 
as of the pancreas. 

Spinal Fluid: 

1. Dilute the spinal fluid 1:5 (1 / 4) with water and use as a protein-free filtrate. 
Divide nominal results by 2. 

2. If the sample contains protein, dcprotelnlze by Da(OH) 2 -ZnS0 4 method and 
proceed as above. 






( 



( 



182 



GLUCOSE TOLERANCE 

References : 

1. Anderson, Q. E, , etal., Am. J. Clin. Nutrition 4, 673 (1056) 

2. Silverstone, F.A, , M. Brandfonbrener. W.W. Shock and M.J. Ylengst, J. CHn. 
Invest. , 36, 504-14 (1967) 

3. Smith, L.E. and Shock, N.W. , J. Gerontology 4, 27 (1949) 

This test determines the ability of the various organs concerned to handle a rapid 
Influx of glucose. The organs Involved include the pancreas (islet cells), the ltver, 
the pituitary, the adrenal cortex, and possibly others. It Is of great value In detecting 
early diabetes. 

Procedure : 

The patient should have been on an average diet for the preceding two or three days. 
He should have fasted for the 12 or 14 hours Immediately preceding the test. He can be 
allowed, and should be encouraged, to drink water enough not to be dehydrated and to 
have an adequate urine flow. 

1. Have patient void. Save specimen, Take a blood sample for glucose determin- 
ation. 

2. Have patient drink a solution of 50 g. glucose dissolved In 300 to 400 ml. of 
water and flavored with the Juice of 1/4 to 1/2 a lemon. Some techniques use doses of 
one gram per kilogram of body weight. 

3. Take blood specimens for glucose determination at 1/2, 1, 2, and 3 hours after 
the ingestion of the glucose, Collect urine specimens at approximately the same time, 

Determine the glucose concentrations In the five blood specimens and test the 
urine specimens qualitatively for glucose. Plot the results. 

Interpretation : 

Blood : There are four criteria which should be considered in the interpretatisr 
of an oral glucose tolerance curve. 

Normal Values 

1. The fasting blood glucose value. 90-120 

2. The highest blood sugar value. 130-140 

3. Time at which the highest value was reached. 46-60 mln. 

4. Time of return to the fasting level. 1 1/2-2 1/2 hra. 

Urine : With a normal renal threshold, not more than a very small amount of 
glucose should appear In the urine. When glucose does appear it gives a rough lndlca- 

183 



tion oi me renal threshold when simultaneous blood sugars are taken Into considera- 
■on. All urine samples giving a positive qualitative test for glucose are pooled and 
ie total glucose determined. 

Intravenous glucose tolerance test; 

This test Is carried out whenever altered Intestinal absorption may require It. In 
the great majority of cases, the oral test is o.uite adequate, 

Procedure: 

One suggested method Is as follows: 

1. The patient Is prepared as outlined above for the oral glucose tolerance test. 

2. Fasting blood and urine samples are taken, 

3. 0.5 g. glucose per kilogram of body weight are given -intravenously In the form 
of a 20% solution over a 5-minute period, 

A fixed amount of glucose may be used with little effect on the results. 

4. Blood specimens are collected from the opposite arm at the end of the injection 
and at the end of the one-hour, two-hour, and three-hour periods following the start 
of the injection. 



Interpretation : Normal Value 

1. Fasting blood glucose value 90-120 (Folin-Wu) 

2. Original level (or lower) reached in 30-60 minutes 

Insulin Tolerance Test: 






This test 1b used to Investigate patients with various endocrine disorders. 

Patient preparation : A diet containing more than 300 g. of carbohydrate daily must 
be taken by the patient for 3 days preceding the test. The test is carried out on the 
fourth day with the patient in a fasting state. 

Procedure : 

1. Blood Is taken for a control fasting blood sugar. 

2. Insulin is injected intravenously (0. 1 units per kilogram body weight). 

3. Additional blood samples are taken for blood sugar at 20, 30, 46, 60, 90 and 
120 minutes after the Insulin Injection. 

Inte rp r etatlon : 

1. The normal response Is an immediate fall (at about 30 minutes) to about 40% of 
the fasting level, followed by a gradual Increase back up to normal fasting values within 
90-120 minutes. There are two abnormal types of response. 

a. Insulin Resistance : This is evidenced by a very slight and delayed fall In 
blood sugar values. This Is seen in some cases of: Diabetes mellitua; Adrenal cortical 
hyperf unction (Cushlngs syndrome); Anterior pituitary hypert unction (acromegaly). 

b. Hypoglycemic Atonia: This is evidenced by a normal fall In blood sugar but 
the subsequent rise does not occur or is delayed. This is seen In some cases of: 
Anterior pituitary hypofunction^Simmond's disease); Adrenal cortical hvpofunetion (Ad- 
dison's disease); Hyperinsullnism. 

184 



< 



5 -HYDROXY- INDOLE ACETIC ACID 
Urine (Qualitative) 

References: 

SJoerdania, A., Welosbach, H. , & Udenfrlend, S: Simple test for diagnosis of 
metastatic carcinoid. J. A.M. A. 159 , 397 (1955). 

Udenfrlend, S. , Welssbach, H. , & Clark, C: The estimation of 5-hydroxy- 
tryptamine (serotonin) In biological tissues. J. Biol. Chem. 215, 337-344 (19S6). 

Udenfrlend, S. , Weissbach, H. , & Titus, E.: The identification of S-I^droxy- 
3-lndoleacetic acid in normal urine, and a method for its assay. J. Biol. Chem. 
216 . 499-506 (1955). 

Principle : 

l-nitroso-2-naphthol in the presence of a small amount of nitrous acid will yield 
a purple derivative with 5-hydroxy-indoles and this reaction is the basis for detection 
of abnormally large amounts of 5-hydroxy-indoleacetlc acid in urine (40 mg. or more 
per 24-hour specimen). False positives are given by p-hydroxyacetanillde which 1b 
found in the urine only after administration of acetanllide drugs. Color formation la 
sometimes inhibited by large amounts of keto acids. 

Reagents : 

1. l-nttroso-2-naphthol (Eastman) - 100 mg. of the compound is dissolved in 100 
ml. of 95% ethanol. 

2. Reagent A - 2. 5 g.- of pure sodium nitrite Is dissolved in 100 ml. of distilled 
water. Keep solution refrigerated. Prepare fresh every 20 days. 

3. Reagent B - 19.6 g. of sulfuric acid (concentrated reagent grade) is weighed 
in a 50 ml. beaker. Add the acid to 65 ml. of distilled water. 

4. Ethylene dlchloride - purified. 

Procedure : 

1. Into a 5 -inch test tube measure 0. 2 ml. of the urine to be tested. Mark this 
tube "T". Into a second 5-inch test tube, marked "C" for control, measure 0.2 ml. 
of a known negative urine. 

2. Add 0.8 ml. distilled water to each tube. 

3. Prepare the Diazo reagent by adding 0. 2 ml. of reagent A to 5 ml. of JTeagent 
B. This solution must be used within thirty minutes. 

4. To both test tubes add 0.5 ml. of l-nltroso-2-naphthol solution and mix well. 

5. To both tubes add 0. 5 ml. of Diazo reagent (prepared in step 3) , and again mix 
well. Allow solutions to stand for five minutes. 

6. Add 5 ml. of ethylene dlchloride to each tube and mix well to extract the excess 
l-nltroso-2-naphthol. If turbidity results, the tubes should be centrlfuged. A purple 
to black color in the upper layer is positive for 5-hydroxy-indoleacetlc acid. A yellow 
color which often appears Is of no significance. A positive test indicates the presence 
of over 8 mg. per liter. 

Interpretation: 

A positive test for 6-hydroxy-indoleacetic acid is comparatively rare. The normal 

185 



24-hour output Is between 2 to 6 mg. When positive, this test Is a specific Indication 
of a metastatic carcinoid condition. In 5 patients with carcinoid syndrome the 24-hour 
output varied from 140 to 628 mg. 
























































186 



( 



ICTERUS INDEX 
Scrum 

Principle : 

The intensity of the yellow color of serum, usually due to bilirubin, is determined 
by comparison with a standard potassium dichromate solution. 

Reagents: 

5% sodium citrate solution for diluting serum. 

Na 3 C 6 H B Cy 2H 2 (U.S. P.) 50 g. dissolve in HgO and dilute to 100 ml. Mix. 

Standards: 

Weigh out 1.000 g. of KJCtJO*. dissolve in water, add 2 drops of concentrated 
H 2 S0 4 , dilute up to 100 ml. , ana mix. Mo clean, dry test tubes place respectively 
0.1, 0.3, 0.5, 0.7, 1.0, 1.2, 1.5, 2.0,- 3.0, 4.0, 6.0, 6.0, 7.0, 8.0, 9.0, and 10.0 
ml. of this solution. Make each volume up to 10.0 ml. with water, and mix. Select 
test tubes, 11 x 100 mm. size, Pyrex, that have uniform diameters, and clean and 
dry tubes. Fill about two-thirds full with the above solutions, and seal off. Label each 
tube according to the number of tenths of a ml. of the 1% dichromate solution used In 
making it, so that the tubes are labeled from 1 to 100 icterus units. Keep these stand- 
ards in the dark while not being used. 

Method : 

Use selected tubes like the above, and of a diameter uniform with the above. Place 

i 

1 ml. of serum without hemolysis In one of these test tubes, and compare with the ser- 
ies of standards. From this preliminary comparison, dilute the serum with 5% sodium 
citrate (if necessary) so that the final comparison is made with an intensity of 15 units 
or less. Taking the dilution into account, calculate the ICTERUS INDEX of the origin- 
al serum. 

Remarks : 

The ICTERUS INDEX gives the number of times the serum would have to be di- 
luted in order for it to match a 1 to 10,000 solution of potassium dichromate (0.01% 
K^CrgO,,). The normal value is 3 to 5 units. Icterus or jaundice is seldom visible in 
the skin or sclera of a patient until the index rises to about 15 units. The level at 
which the skin changes color is largely dependent on the amount of diffusible bilirubin 
present, while the non-diffusible form has little effect on skin color. 

Even traces of hemoglobin make it difficult or impossible to obtain a match with 
the standards. Llpemia also Interferes; so the patient should be In a fasting condi- 
tion. Occasionally carotenemla contributes to the amount of yellow color in the serum. 



187 



IODINE PHOTE IN-BOUND 
Scrum 



( 



References 



1. Thompson, H.L. , Klugerman, M.R. and Truemper, J., "A method for pro- 
tein-bound iodine: the kinetics and the use of controls in the ashing technique, " 
J. Lab. and Clin. Med, . 47, 149-63 (1946). 

2. Barker, S.B. , Humphrey, M.J. and Soley, M.H. , "The clinical determination 
of protein-bound Iodine," J. Clin. Invest. , 30, 55-62 (1951) 

3. Klugerman, M.R. , "A simple and rapid calculation in Barker's method for 
blood-protein-bound Iodine," Am. J. Clin. Path. , 24, 490-495 (1954). 

Principle: 

The estimation of serum protein -bound iodine (PBI) has become an important tool 
In confirming the existence of thyroid dysfunction. Since its introduction the PBI deter- 
mination has been proven to be a more reliable and reproducible indicator of thyroid 
function than the basal metablollc rate (BMR) although it, too, has its own pitfalls. 

In the dry-ashing sodium carbonate method of PBI determination, the Iodine is iso- 
lated from the serum by precipitation and washing of protein followed by drying and in- 
cineration. These steps eliminate constituents that would interfere with the colorlmetric 
determination of iodine. The iodine concentration in the ash la then determined. 

The colorlmetric technique for the determination of trace quantities of Iodine is 
based on the catalytic action of Iodide on the cerlc-arsenite system. 

2 Cer' 4 / As^ 3 > 2 Ce# / As^ 

A definite linear relationship between reaction time, i.e. , the rate of decolorization 
of the eerie ion, and the iodide concentration was shown. Therefore, by determining 
the cerlc Ion concentration at the end of a given period of time the iodide concentra- 
tion may be calculated. The cerlc ion concentration Is determined photometrically. 



Apparatus : 

1. Photoelectric colorimeter: Any of the standard instruments. may be used for the 
PBI determination If a proper range has been ascertained by estimating the optimum 
concentration of eerie ion for the ceric-lodlde-arsenlte system. 

2. Cuvets 



( 



188 



3. Muffle furnace 

4. Drying oven 

5. Heavy wall glass Ignition tubes (25 x 200 mm.) 

ReagentB 

1. Zinc Sulfate : dissolve 100 grams ZnS0 4 '7H„0 la water and dilute to one liter. 

2. Sodium Hydroxide (0. 5N): Dissolve 20 grams NaOH In water and dilute to one 
liter. Note: The zinc sulfate and sodium hydroxide solutions should be so adjusted that 
when 10. ml. of the zinc sulfate is diluted with 50 to 70 ml. H„0 and titrated with 0, 5N 
NaOH, about 10.8-11.2 ml. of the NaOH will be required to produce a'falnt permanent 
pink color with phenolphthaleln. 

3. Hydrochloric acid (2.0 N): Dilute 170 ml. analytical reagent grade concentrated 
HC1 to one liter with H z O. Titrate against 1.0 N NaOH. 

4. Sulfuric acid (7N): Dilute 200 ml. concentrated H 2 S0 4 to one liter with H„0. 

5. Sodium Carbonate (4, N): Dissolve 212 grams of Na 2 COo (analytical reagent) 
in HgO and dilute to one liter. Titrate against standard 1.0 N HC1 with methyl orange 
Indicator. 

6. Cerlc Sulfate (0.012 N): Dissolve 8.02 grams of eerie ammonium sulfate, 
(Ce(SO )--2(NH 4 ) 2 S0 4 • 4H 2 0) in a one-liter flask containing 500 ml. H 2 and 230 ml. 
7 N H So , When the solution is clear, dilute to the liter mark with H 2 0, 

7. Arsenious Acid (0. 012 N): In a one Liter flask dissolve 5. 84 grams of As-O, la 

30 ml. of 1 N NaOH, warming to hasten solution. Add 300 ml. HgO and dilute H.SO. until 

the solution Is slightly acid to litmus. This requires approximately 4.5 ml. of the 7 N 

HoSO. . Dilute with H o to the liter mark. 
& 4 & 

8. Iodide Stock Standard : Dissolve 118.1 mg. Nal, analytical reagent, or 130.8 mg. 
KI, la H 2 and dilute to one liter (1 ml. = 100.0 micrograms I"). Dilute stock is ob- 
tained by diluting 4 ml. of the above stock to one liter with H 2 0. (1 ml. = 0.40 ug l~). 
Working Stock is obtained by diluting 10 ml. of dilute stock to 100 ml. with H 2 0. 

(1 ml. =0.04 micrograms V). All of these solutions have been found to keep indefin- 
itely at refrigerator temperatures, ft Is preferable, however, to prepare a new work- 
ing standard every week or two. 

Analytical Procedure 

A. Precipitation and Washing of Protein 



1. Add 10.0 ml. H 2 to each ignition tube. 



189 






2. Pipot 1.00 ml. serum Into each tube. 

3. Add 1.0 ml, of the ZnSO recent and mix well, 

4. Add 1.0 ml. of 0.5 N NaOH and mix thoroughly with a stirring rod. 

5. Centrifuge for 10 minutes at 3500 rpui. 

6. Pour off and discard the supernatant fluid. 

7. Add 10.0 ml. HgO to each tube and mix. 

8. Centrifuge and discard supernatant as above, 

9. Wash at least one more time, 

10. Add 1.0 ml. of 4.0 N Na„CO„ solution, Do not mix. 

B. Drying and Incineration 

1. Place the ignition tubes in the drying oven (85 to 95°C.) to drive off the water. 
This usually takes about 12 hours. 

2, After drying, place the tubes in the muffle furnace (600 £ 25°C.) for three hours. 

C, Colorlmetrlc Determination 

1. Add 2 ml. of 2 N HC1 to the ash. Tilt the tubes at a 30° angle so that no material 
Is lost in the effervescence. Allow to stand for 10 minutes. 

2. Add 1 ml. 7 N H 2 S0 4 and 3 mis. HgO. 

3. Add 0.5 ml. of the arsenious acid solution and mix well by shaking. 

4. Add 1 ml. of the eerie sulfate reagent, at 30 second interv; Is, to the samples 
being tested, and mix well by shaking. 

5. Place each tut/e In a 37°C. water bath Immediately after the addition of the 
eerie sulfate. 






6. Set the photometer at 100% transmittance using a wavelength of about 420 mu 
with a distilled water blank and at 0% transmittance with a reference blank (1 ml. cerlc 
sulfate solution In 6.5 mis. HO). Recheck these setting several times in alternation. 

7. Read the transmittance of the first sample exactly 12 minutes after the addition 



190 






of the eerie sulfate solution, pouring the solution from the fgilftton tubes into the appro- 
priate cuvcts, Continue reading each of the other samples at 90 second Intervals corres- 
ponding to the time of addition of the ccrlc sulfate solution,- each one being read 12 
minutes after the cerlc sulfate addition. 

TABLE 1 
SUMMARY OF PROCEDURE 





Serum 
Sample 


Reagent 
Blank 


1. H 2 


10.0 ml. 


— 


2. Serum 


1.0 ml. 


— 


3. ZnS0 4 


1.0 ml. 




4, O.BNNaOH 




... 



5. Centrifuge and discard supernatant, wash with water 2 times 

6. 4.GNNa 2 C0 3 1.0 ml. 1.0 ml. 

7. Dry In oven and incinerate In muffle furnace 

8. 2 N HC1 (let sit 10 minutes) 2. ml. 2.0 ml. 



9. 7 N HjjSO^ 



1.0 ml. 1.0 ml, 



10. h 2 3.0 ml. 3.0 ml. 

11. Arsenlous acid 0.5 ml. 0.5 ml. 

12. Ceric Sulfate 1.0 ml. 1.0 ml. 



Calculations 

A standard curve is determined using the prepared Nal or KI standard solutions. 
From this curve the iodine concentration at a given tranamlttance may be determined. 



191 



The solutions of varying Iodide concentration for tho establishment of the standard 
curve arc prepared In live following maimer. 

1. Add the appropriate amount of Nal or KI standard solution and water to each 
cuvet (see Table H), 

2. Add 1.0 ml. of 4.0 N Na CO, solution to each tube. 

3. Add 2/0 ml. of 2.0 N HC1 with the tubes tilted at a 30° angle, and let them sit 
for 10. minutes. 

4. Add 1,0 ml. of 7.0 N H 2 S0 4 . 
6. Add «0.5 ml. of arsenlous acid, 

6. Add 1.0 inl. of cerlc sulfate solution to each cuvet at 30 second intervals; mix, 
place the tubes in the water bath; read the light transmission in the same manner as 
described for samples under Analytical Procedure. 

7. The standard curve is determined by plotting per cent tranemittance against 
the iodide concentration, on semi -log paper. 









Iodide ■Concentration 





(ug/ml) 




■ 


0.00 
0.02 






0.04 
0.06 






0.08 
0.10 






0.12 
0.14 
0.16 
0.18 
0.20 




Notes 







TABLE II 












KI or Nal 
(ml) 




H 2<L 
(ml) 


0.0 




2.0 


0.2 




1.8 


0.4 




1.6 


0.6 




1.4 


0.8 




1.2 


1.0 




1.0 


1.2 




0.8 


1.4 




0.6 


1.6 




0.4 


1.8 




0.2 


2.0 




0.0 



Standards: A standard should be run with each group of samples to serve as a 



192 






check on the method, accuracy and equipment. Either of two types of standard may be 
used: (1) the serum standard which Is a commercially prepared serum of known protein- 
bound iodine concentration (e.g. , Versatol by the Warner -Chi Icott Laboratories of 
New York) or (2) the prepared standards of the Nal and KI standard solutions. 

The serum standard Is run in the same manner as the serum sample with the ex- 
ception that the standard serum Is used Instead of the unknown sample. 

When Nal or KI solutions are used as standards the precipitation steps of the anal- 
ytical procedure are omitted. One-half ml. of the standard working Nal or KI solution 
Is placed In an Ignition tube, and one ml. of the Na-COg solution is added. The tube is 
then carried through the' remaining steps of the analytical procedure along with the 
group of samples. 

Blanks ; A reagent blank should also be run with each determination. This consists 
of adding 1. ml. of the Na CO- solution to a dry ignition tube and carrying the tube 
through the remaining steps In Die analytical procedure along with the group of samples. 
Any contamination occurring in the determination will be shown by the blank. 

Contamination; The reagents and glassware may be very easily contaminated by 
Iodine or mercury (which inhibits the reaction) due to the very small amounts being 
determined. Scrupulous care should be taken to guard against this possibility. It is 
recommended that a separate room be used for the PBI determinations. 

Distilled water : The water used in this determination should be glass redistilled 
from an alkaline solution. 

Interpretation 

The PBI Is an important clinical and diagnostic tool when used with knowledge of its 
applicability and limitations. It is more often compatible with clinical and pathologic 
evaluation of thyroid disorder than the BMR since it is a direct measurement of circu- 
lating calorlgenic substance while the BMR is an indirect Indicator of thyroid function 
and may be affected by many non-thyroid hypermetabollc and hypometabolic diseases 
or conditions, as well as by emotional attitude and the responses and level of external 
stimuli. 

The normal PBI concentration in the human body has been found to be 3. 5 to 8. ug 
per 100 ml. The PBI is generally elevated in hyperthyroidism and falls after thyroid- 
ectomy or treatment with radioactive iodine or antithyroid drugs. In cretinism or 
hypothyroidism the PBI is generally below normal. However, in some borderline cases 
r when the patient has hyperthyroidism or hypothyroidism the PBI may be Just within 
the normal range. In thyroid carcinoma and thyroiditis values for the PBI follow no 
consistent pattern. 



193 



Nonthyroid diseases, especially neuroses, psychoses, cardiovascular-renal 
diseases, non-thyroid endocrine disturbances, blood dyscraslas, ext rathy rot d malig- 
nancy, hepatic and biliary tract diseases, infectious diseases and dermatoses seldom 
cause any variation in the PBI. 

The major difficulty in the determination of the PBI is that it is greatly affected by 
exogenous Iodine In any form. Therefore the thyroid status of the patient cannot be de- 
termined when the patient has bee: receiving Iodine medication or any drug or treat- 
ment involving Iodine or bromine. 

Organic Iodine -containing con p Minds known to affect protein -bound Iodine determin- 
ations include Iodine -containing r* U opaque dyes used in angiocardiography, cholecysto- 
graphy, urography, bronchography , or myelography; lodlnated amebicldes (chlnlofon, 
lodochlorhydroxyquln, and dllodoH uroxyquln) ; vaginal suppositories such as Floraquln, 
which contains diiodohydroxyquln; penicillin O diethylamlnoethyl ester hydriodlde (Neo- 
penil); and lodothlouracll. 

Preparations containing inorgsnlc Iodine include Lugol's solution, tincture of Iodine 
and syrup of hydrlodlc acid. Certs in vitamin and cod liver oil preparations may also be 
rich In iodine, while barium sulfat ) Is said to give rise to elevated protein-bound iodine 
due to contamination iodine. Sulfo iromophthaleln (Bromsulphaleln) though not con- 
taining iodine, also causes appare it elevation of plasma protein-bound iodine. 









( 












194 



iron 

Serum 

Reference : 

Fister, H. J. , "Manual of Standardized Procedures for Spectcophotometric Chem- 
istry." Standard Scientific Supply Co. , New York City, N. Y. (1950). 

Principle : 

A sample of plasma or serum is incubated with hydrochloric acid. The protein Is 
then precipitated and the ferric ion (Fe^/) i s reduced to the ferrous state with hydro- 
quinone. The ferrous ion is then reacted with a-a 1 dipyridyl to form an intense red 
colored complex (Blau reaction) . 

Apparatus : 

1. Ostwald-Folin pipet accurately calibrated to contain 4. 00 ml, 

2. Coleman spectrophotometer, with small cuvets. 

Reagents : 

1. Water, distilled, iron-free: Redistill distilled water In an all-glass pyrex dis- 
tillation apparatus until iron-free. 

Test for the presence of iron: Treat 10 ml. of water with 1 ml. concentrated 
hydrochloric acid, 0.1 ml. concentrated nitric acid, 4 ml. 3 N potassium thiocyanate 
and 2 ml. iso-amyl alcohol. Shake thoroughly and allow the alcohol layer to separate: 
a colorless alcohol layer Indicates the absence of iron. Use this test for all reagents 
and standards and-where indicated in the test procedure. 

2. Hydrochloric acid 0.3 N. Add 30 ml. of concentrated hydrochloric acid to 1000 
ml, of water. 

3. Trichloroacetic acid 20 per cent aqueous. Add 20 g. of trichloroacetic acid to 
100 ml. volumetric flask and dilute to mark with water. Mix well. 

4. Sodium acetate, saturated aqueous. Place 130 g. of sodium acetate, crystal- 
line (NaC 2 H 3 02'3H 2 0) into a bottle and add 100 ml. of water. Note: complete satur- 
ation is essential, excess crystals should be present. 

5. Hydroqulnone , 1 per cent aqueous. Dissolve 0.5 g. in SO ml. of water. This 
solution must be made fresh each day. Ascorbic acid may be substituted for hydroqulnone. 

6. a-a' dipyridyl, 0.2 per cent aqueous. Dissolve 200 mg. in 10 ml. of water 
and dilute to 100 ml. Reagent must be colorless. Discard if colored. Keep In brown 
glass bottle in refrigerator. 

7 . Iron Standard: 

(a) Iron Stock standard: (1 ml. contains 1 mg. of ferric Ion). Thoroughly 
clean a small piece of iron wire of known iron content (special for standardization, 
usually 99.85 per cent Fe) with fine emery cloth or paper until shining and free of 
rust and dirt. (A sample of Ingot iron may be used. This may be procured from 
National Bureau of Standards, Washington, D.C.) Knowing the iron content, weigh 
out an amount of wire that contains 100 mg. of Iron. Into a 250 ml. flask place 10 ml. 



195 



of concentrated nitric acid and approximately 40 ml. water. Heat to boiling (gently) 
and add wire, boll (gently) until completely dissolved and one minute longer, cool and 
transfer to a 100 ml. volumetric flask, dilute to the mark with water. 

<b) Dilute standard: (1 ml. contains 0.01 mg. Fc): Place 1.00 ml. of stock 
standard in 100 ml. volumetric flask and dilute to (he mark with water. 

Procedure : 

1. Into a 15 ml. centrifuge tube, place: 

2 ml. of HC1 0.3 N and 4.00 ml. of serum or plasma (rinsing out pipct several 
times In the tube.) Mix well, especially notLng I be tip of the centrffugo tube. 

2. Place In water bath for one hour at 37° C. 

3. Add 2 ml. of 20% trichloroacetic acid dropwlBe with shaking. 

4. Stopper and shake tube vigorously. 

5. Allow tube to stand at room temperature for one hour. 

6. Centrifuge at liigli speed (2000 rpm) for 15 minutes. 

7. Into a small cuvet place 4.00 ml. of the clear supernatant fluid. 

8. Into another cuvet place in order: (Reagent blank.) 
2.00 ml, of water 

1.00 ml. 0.3 N HC1 

1.00 ml. trichloroacetic acid 20% 

Mix well 

9. To each cuvet add In order: 

0.5 ml. saturated sodium acetate 
0.3 ml. hydroqulnone 
1.00 ml. a-a* dipyridyl reagent 
Mix well 

10. Allow to stand for one hour at room temperature. 

11. Read transmlttancy of the sample against the reagent blank set at 100 per cent 
transmittance at 520 mu. 

12. Read the concentration from the calibration curve or calculate by comparison 
with a standard run simultaneously. 

Calibration 

In a series of seven accurately calibrated 10 ml. volumetric flasks place respec- 
tively 0,1,2,4,6,8,10 ml. of the dilute standard (0.01 mg./ml.) and dilute to volume 
with Iron-free water. 

These standards represent concentrations of iron equivalent to 0, 0. 1, 0.2, 0.4, 
0.6, 0.8, 1.0 mg. per 100 ml. of sample. (Prepare just before use). 

Transfer 2 ml. of each, calibration standard to a correspondingly marked cuvet. 
To each cuvet, add In the order named, mixing after each addition. 
1 ml. 0.3 N HC1 
1 ml. trichloroacetic acid 20% 
0.5 ml. saturated sodium acetate 
0.3 ml. hydroqulnone 
1 ml. a-a' dipyridyl 
Allow to stand at room temperal"re for one hour. Set the zero concentration 

196 



standard at 100 per cent transmlttance at a wavelength of 520 mu, and record the 
transmlttancy of the 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mg. per cent standard b. (The 
transmlttancy of the 1.0 mg. standard Is approximately 26 per cent.) 

Plot the observed values on semi-log graph paper (or convert to optical density 
and plot on linear paper). (The points fall in a straight line which passes through the 
origin.) 

Use this graph to find the concentrations of unknowns. 

Notes : 

1. Plasma or serum from blood must be withdrawn during postabsorptive state: 
The following precautions should be carefully observed to prevent hemolysis. 

(Slight traces are of no consequence but samples showing gross hemolysis cannot be 
used.) 

Withdraw blood with minimum stasis, using absolutely dry syringes and stain- 
less steel needles (gold or platinum needles are preferable) and allow the syringe to 
fill without exerting suction with the plunger, Remove needle from syringe and allow 
blood to flow slowly down the side of the collection tube without pressure on the plunger. 
For plasma analysis use sodium citrate as anti-coagulant. (Fluorides, oxalates and 
tungstates interfere.) Analysis is preferably made on serum. The plasma or serum 
must be separated from the red cells within one hour after the collection of the sample . 

2. It Is advisable to run a standard along with each determination Instead of relying 
upon the constancy of the calibration curve. 

Interpretation: 

The normal ranee of values is: 0. 08 to 0. 18 mg. serum iron per 100 ml. serum. 
Increased values are seen In hepatitis, hemochromatosis and pernicious anemia, 
Decreased values are seen in the Iron deficiency anemias. 












197 



IltON -BINDING CAPACITY 
Blood 



Reference : 

Varley, Harold, "Practical Clinical Biochemistry", Intersclence Publishers Inc. , 
N. Y. , N. Y. ; page 324 (1954). 

Ventura, S. , Determination of the unsaturated lron-blndlng capacity of serum, 
J. of Clin, Path. 5, 270-274 (1962). 

Principle : 

The iron in the plasma Is bound to a specific iron-binding component of the beta- 
globulin, only part of which Is saturated In normal plasma. The iron protein complex 
is red, so that it Is possible to measure the unsaturated iron-binding capacity, that la, 
the amount of iron which can be taken up by this serum globulin, A solution of an iron 
salt Is added to the serum, and the increase in red color Is read on the Beckman 
Spectrophotometer. From this reading the per cent of saturation and the Total Iron 
Binding Capacity may be found If the results of a Serum Iron determination are also 
known. 

Apparatus : 

1. Beckman DU Spectrophotometer with four quartz cuvets. 

Reagents : 

1. iron Standard : 35. 2 mg. of reagent grade ferrous ammonium sulfate, 
Fe(fIH 4 ) 2 (S0 4 )2 , 6H 2 O l and^20 ml. of 1 N acetic acid is diluted to 1000 ml. using 
iron-free, triple distilled water. Mix well. This gives a standard with a concentra- 
tion of 5 micrograms of Iron per ml. of solution. 

2. Sodium Chloride Solution: 0. 85 gm. of pure, dry sodium chloride is added 
to a 100 ml. volumetric flask and diluted to the mark with triple distilled water. 

3. Water: The water used In every step of this determination should be triple- 
distilled and iron-free, preferably given a final distillation in all glass apparatus. 
Since the color Is very sensitive In this test, even trace quantities of Iron In the water 
will profoundly alter results. 

Procedure : 

1. A 20 to 25 ml. specimen of clotted blood Is required; at least 6 ml. of serum 
Is required for the test. 

2. Using fllx test tubes, which have been rinsed at least 5 times with iron-free 
water, number them B, 1, 2, 3, 4, and 6. Add exactly 1.0 ml. of clear, unhemolyzed 
serum to each tube. 

3. To each tube add exactly i.Sml. of sodium chloride solution. Mix thoroughly 
with serum. 

4. Add to the tubes the proper proportions of sodium chloride and Iron standard 
as given In the following table: 



108 



( 



< 



< 



Iron St.iintln.rd 


Sodium Chloride 


0.0 ml. 


1.0 ml. 


0,2 ml. 


0.8 ml. 


0.4 ml. 


0.6 ml. 


0.6 ml. 


0.4 ml. 


0.8 ml. 


0.2 ml. 


1.0 ml. 


0.0 ml. 



Tube 

B 
1 
2 
3 
4 
5 

5. Stir the contents of each tube well with stirring rods. Allow to stand six min- 
utes for full color development. 

6. Transfer the solutions to quartz cuvets for reading on the Beckman DU. Read- 
ings are made at 520 mu using tube B for the blank. The serum has become saturated 
when the optical density has reached a maximum. 

7. The number on the first tube to reach saturation is multiplied by 100 to give the 
amount of iron in micrograms per cent needed for saturation. When Total Iron-Binding 
Capacity Is requested, this result Is added to the results of a serum Iron determination 
on the same sample of serum. 



Example : The optical density readings on a given sample of serum were found to 
be: 

1. - 0.40 

2. - 0.56 

3. - 0.65 

4. - 0.64 

5. - 0.65 

Tube #3 is the first tube to reach saturation, hence the number 3 is multiplied 
by 100. 

3 x 100 = 300 micrograms % (0.3 mg.%) 

To calculate the total Iron-binding capacity, this result is added to the results 
of a previous serum iron which was found to be 0. 16 mg. %. Thus: 

0.16 {Serum Iron) 

0.3 (Iron-binding Capacity) 

0.46 mg.% (Total Iron Binding Capacity) 

Interpretation : 

Following are accepted normal values for the normal adult male and female, given 
in mg. per cent. 



199 



Normal Male Normal Female 

Iron -Binding 0.150 - 0.222 0.144 - 0.322 

Capacity (Aver. 0.205) (Aver. 0.104) 

% Saturation 30% - 44% 30% - 44% 

(Aver. 34%) (Aver. 33%) 

Total Iron-Binding 0^254 - 0.432 0.224 - 0.415 

Capacity (Aver. 0.311) (Aver. 0.288) 

In Iron deficiency, while serum Iron is lowered, there Is an Increase above nor- 
mal in both the unsaturated iron-binding capacity and total carrying capacity of the 
serum. This 1b in contrast to infection in which serum Iron is similarly reduced, 
but where the iron-binding capacity and total capacity are reduced as well. A high 
serum iron and high percentage of saturation of the iron-binding protein Is found in 
refractory anemia, pernicious anemia, hemochromatosis, liver disease, and trans- 
fusion hemosiderosis. 









< 















200 






Reference b: 



NEUTRAL 17^KETOSTEU01DS 
Urine 






(1) Callow, N., Callow, R. , Emmcns, C: Colorlmetrlc determination of sub - 
stances containing the grouping -CII„*CO- in urine extracts as an Indication of andro- 
gen content. Btochem. J. 32, 1312-1331 (1938). 

(2) E tig strom, W. , Mason, H. : A study of the colorlmetrlc assay of urinary 
17-ketosterolds. Endocrin. 33, 229-238 (1943). 

P rinciple: 

The 17-ketosteroida are separated from theirwater soluble conjugates by hydroly- 
sis with concentrated HC1, They are then extracted from the hydrolyzed urine with 
ethyl ether. The ether extract is washed with sodium hydroxide and distilled water to 
remove all phenolic and acidic impurities, and finally evaporated to dryness. The rest- 
due is taken up in absolute ethanol. An aliquot of this alcofiolic urine extract la treated 
with potassium hydroxide and m-dinitrobenzene to develop a red-green color which is 
compared with a standard in the spectrophotometer. 



. 









Apparatu s: 



1. Coleman Junior Spectrophotometer, Model 6A 

2. 12 matched round cuvettes, 19 x 150 mm. 
Reagen ts: (All chemicals used should be of Reagent Grade) 

1. Hydro chlo ric A cid: concentrated analytical grade 

2. Ethyl Ether: peroxide -free 












3. 2N S odium Hydroxide S olution: 80 grams of NaOH pellets are dissolved In 

500 ml. distilled water In a 1 liter Pyrex volumetric flask, and diluted to the mark when 
cool. Preserve In al liter polyethylene bottle. 

4. Ethanol: Dehyrated, N.F. 

5. m-dinltroben7.ene: The Reagent Grade compound should be recrystalllzed once 
from ethyl alcohol and carefully dried before using. The purified compound should be 
stored in a tightly stoppered amber bottle in a cool, dark place. For use In this proce- 
dure, 0.50 grams of the purified crystals are dissolved in exactly 25 ml. of dehydrated 
ethanol to make a 2% alcoholic solution. The solution when stored in an amber bottle at 
room temperature will keep for about 30 days. 



201 






6. 8N Potassium Hydroxide : -15 grains of the Kcagcnl Grade pellets are dissolved 

75 ml. of distilled water and then diluted up to the 100 ml. mark In a volumetric flask. 
The solution Is cooled to 15°C. in the refrigerator and then diluted to the mark. The 
normality should be checked by titration with a primary standard. The solution should be 
stored in a polyetliylenc bottle and the litre of the solution checked and corrected every 30 
days. 

7. Diluting A lcoho l: '100 ml. of 95% alcohol is placed in a 500 ml. graduate and 
diluted with distilled water to the 500 ml. mark.. Store in a glass stoppered bottle. 

8. Dehy dr oisoandroster one Standird: 25 mg. of the steroid Is dissolved in exactly 
25 ml. dehydrated ethanol and stored in a glass stoppered bottle in the refrigerator. 
This is the stock solution with a concentration of 1 mg./ml. The working standard Is 
made by carefully diluting 2 ml. of the stock standard with 6 ml. of dehydrated ethanol 
In a 10 ml. graduated cylinder and mixing well. This gives a working concentration of 
0. 25 mg. per ml. The working standard is stored at room temperature. 

(Pure dehydroisoandrosterone may be purchased from Sigma Chemical Co. , 4648 Easton 
Ave., St. Louis 13, Mo.) 

Procedure: 



1. Accurately measure and record the total volume of the 24 hour specimen. 

2. In a 250 ml. Erlenmeyer flask, place a 100 ml. aliquot of the urine. Add 15 ml. 
of concentrated hydrochloric acid and two hard glass beads. Place a funnel In the neck 
of the flask to prevent splattering, and bring the contents of the flask to a boil on a hot 
plate. Continue to boll GENTLY for exactly ten minutes. 

3. Immediately cover the flask with a 50 ml. beaker after removing flask from heat, 
and place in refrigerator until urine reaches room temperature or below. 

4. Place cooled urine in a 250 ml. separatory funnel and extract with one 100 ml. 
and two 50 ml. portions of ether. Combine the ether extracts. Discard extracted urine. 

5. Wash the combined ether extracts with 15 ml. portions of 2 K NaOH until the 
aqueous phase is colorless (usually three washings is sufficient). Discard washings. 

6. Wash the ether extracts with three 15 ml. portions of distilled water. Discard 
washings. 

7. Evaporate the ether extract in a 250 ml. glass evaporating dish to dryness in a 
rapid stream of air in the hood. Under no circumstances should heat be applied to hasten 
the evaporation of remaining traces of water. 

8. Using a glass rod with a rubber policeman, the residue is taken up quantitatively 
in exactly 2 ml. of dehydrated alcohol, and stored temporarily in a glass stoppered 5 ml. 
g raduate . 

202 



9. A test tube rack is setup with 19 x 150 mm. cuvotles: one cuvette marked 
"RB" for Reagent blank, one murked "S" for Standard. Two cuvettes are required 
for each sample to be run: one marked "T" for Test and one marked "U" for urine 
blank. A Urine Blank is run to correct for extraneous color from urinary chromogens. 
Using 0.2 ml. pipettes (Serological), add the following reagents to the appropriate 
cuvettes in the exact order given: 

TEST "T" (One for each sample) 



0.2 ml. alcoholic urine extract 
0.2 ml. 2% m-dinitrobenzene 
0.2 ml. 8NKOH 












URINE BLANK "U" (One for each sample) 

0.2 ml. ethanol 

0_2 ml. alcoholic urine extract 

0.2 ml. 8NKOH 

REAG ENT BLA NK "RB " (Only one required) 
0.2 ml. ethanol 
0.2 ml. 2% m-dinitrobenzene 
0.2 ml. 8NKOH 

STANDAR D "S" (Only one required* 

0.2 ml. dehydrolsoandrosterone standard soln. 
0.2 ml> 2% m-dinitrobenzene 
0.2 ml. 8N KOH 

10. Mix the contents of each cuvette well. Place all tubes in a dark place at room 
temperature, allowing exactly 20 minutes for color development. At the end of this 
time add 10 ml. of diluting alcohol to each tube using a 10" ml. volumetric plpet.. 

11. Read all tubes within 7 minutes in the Coleman Spectrophotometer set at 520 mu. 
The instrument Is first set to read 100% transmission using the Reagent Blank and the 
Standard and all Tests "T" read as % transmission against it. Next the instrument Is 
reset at 100% transmission using distilled water, and all Urine Blanks "U" read. 

Calculation s: 

1. Convert all readings in % transmission to their corresponding optical density 
from an appropriate conversion chart. 

2. To calculate the amount of 17-ketosteroids as mg%; Substitute readings in 
optical density in the following formula: 



203 



Concentration = lQP°LIP-5. t Jl T ") - (OP^JJrineBlanknPl. x 5 

(OD of the Standard "S") 

The factor 0. 5 In the above equation Is derived from the Standard Photometric 
Equation (p. 57). 

3. Total Ketosterold Content of 24 hour specimen: Substitute appropriate values 
In the following equation: 

c onc^Jn_mg% x Total Vt , lume ln ml8 ^ per 24 hour y^um^ 

100 

Example : A 24 hour specimen submitted was measured and found to have a 
Total Volume of 3590 ml. Readings on the Spectrophotometer were found to be: 

Test "T" - 73.2596 (OD =. 0.1352) 

Urin e Blank "U" - 97.50% (OD = 0.0110) 

Stand ard - 48.50% (OD = 0.3140) 

Substituting In the first equation (Step 2) the concentration in mg.% was found 
to be 0.20 mg.%. 



( 






0.1352 - 0. 0110 x .5 = 0.20 mg.% 
0.3140 

To calculate Total Ketosterold Content, the concentration ln mg.% was divided 
by 100 and multiplied by the total volume of the 24 hour speclmsn ln mis. and 
found to be 7. 11 mg. per Total Volume. 

4. The results of analysis are reported in the following manner: 

Total Volume cc 

17 Ketosterolds mg. % 

Total 17 KetOBterolds mg. /T. V. 

Notes: 

In order to constantly produce accurate results with this procedure, instructions 
should be very carefully followed. Slight errors can often produce abnormally high or 
low results. 

The greatest errors in hydrolysis and extraction may be due to: 

1. Too vigorous boiling during hydrolysis, or boiling longer than the prescribed 
10 minutes. THIS I S ONE OF THE BIGGEST SOURCES OF ERROR. 



204 



( 






2. Failure to completely remove phenolic and acidic impurities in the ether extract 
with 2N NaOil. The aqueous phase should be completely colorless during the last wash- 
ing. 

3. Failure to wash the last traces of NaOH from the ether extract with distilled 
water. 

4. Failure to take up the residue from the ether extract in ethanol completely. 



The most sensitive part of the procedure is in color development, and is the section 
in which most of the errors are made. Majdr sources of error include; 

1. Inexact preparation of reagents; especially with 8N KOH. The normality of the 
KOH should be as nearly 8.000N as is possible, and the titer should be checked fre- 
quently - at least every 30 days. 

2. Failure to allow all reagents to reach room temperature before beginning color 
development. 

3. Improperly cleaned cuvettes. Cuvettes should be washed thoroughlyj rinsed at 
least 3 times with tap water and finally, 3 times with distilled water. They should be 
thoroughly dry and free from dust and lint before using. 

4. Inexact color development time. Color development should be exactly 20 min- 
utes from the time 8N KOH is added to the cuvettes until diluting alcohol is finally added 
before reading. 

5. Standard "S" Reading: All results depend greatly for their accuracy upon the 
reading of the standard. The standard MUST read from 45.00% to 51.00% transmission; 
48.50% optimum. Whenever the Standard is not within this range, all cuvette solutions 
should be discarded and the whole color development procedure repeated, using clean 
cuvettes. 

I nterpreta tion; 

17-Ketosterolds in varying quantities are normally excreted in the urine of all indi- 
viduals, the amount depending upon age and sex. Normal 24 hour values include: 

Adult Male 10 to 22 mg. 

Adult Female 6 to 18 mg. 

Children (under 5) 1 to 3 mg. 

Children (5 to 12 yrs.) 2 to 6 mg. 

This test serves as a valuable index of testicular and adrenocortical activity, and 



205 



certain diseases cause an abnormally high or low output. Disorders of the testes, 
adrenal cortex, and anterior pituitary can profoundly affect 17-lcetostcrold excretion, 
rising as high as 800 mg. per diem in rare cases of masculinizing tumors of the 
testes, or dropping as low as 0.2 mg. per diem In Addison's disease or Panhypo- 
pituitarism. 



( 



206 



LIPASE 
Of PttttcimMUs 

References : 

1. Cherry, I. S. , and Crand&ll, L. A. , Am. g, Physiol. 100, 2fi6 (1082) 

2. Henry, R. J. , Sobel, C. , and Bcrkman, S. , Clin. Cham, 3, 77 (11*67) 

3. Goniori, G, , Am. J. Clin. Path. 27, 170 (14)57) 

4. Bunch, L. D. , and Emerson, R. L. , Clin. Chom. 2 t 7B (1956) 

5. Archibald, R. M. , J. Biol. Chcm. 165, 443 (1946) 

Princip le: 

Serum ie incubated with s n olive-oil emulsion substrate. Lipase activity results 
in splitting of the glyceryl -fatty acid ester bond with the liberation of free fatty acids. 
The amount of action is determined by titrating the liberated fatty acids with standard 
alkali using thymolphthalein as the indicator. A glass electrode pH meter may also 
be used to detect the end-point. 

Reagents : 

1. Olive-oil emulsion Olive oil may be purified as follows: 

Gum acacia 12.5 g Wash 100 ml. of olive oil with thorough 

Olive oil, 50 ml. shaking in a Beparatory funnel, once with 

Distilled water 100 ml. 100 ml. of 0.5% NaHCOg and then twice 

Sodium benzoate 0, 2 g. wi)th water. 

Dissolve the gum aca< ia and the sodium benzoate in the water. Add the olive oil 
and emulsify by passing the mixture through a hand homogenlzer until a smooth emul- 
sion results, usually 3 to 5 times. Store in the refrigerator. Shake well before using. 
Preferably pass the emulsion through the homogenlzer just prior to use or prepare 
fresh each day. 

As an alternate preparation method, emulsify at high speed for 15 minutes in 
a Waring blendor. 

2. Phosphate buffer 0. 67 M pH 7. 5 

Na 2 HP0 4 -7H 2 15.7 g. 
KH„P0 4 anhyd. 1. 2 g. 
Dissolve the salts in distilled water and dilute up to about 1 liter. Add one ml, 
toluene and keep in refrigerator. 

3. Alcohol -ether mixture. 

Add 9 parts (by volume) of 95% ethyl alcohol to 1 part dicthyl-ether. 

4. Thymolphthalein indicator 

0.4 g. of the indicator are dissolved in 50 ml. 95% ethyl alcohol and then 
50 ml. of distilled water are added. 

6. 0.05 N NaOH. The precise normality must be known. 

Dilute 5 ml. of 2.5 H NaOH up .to 250 ml. and standardize against standard 
acid using thymolphthalein an the indicator. 



207 



Procedure ; 

1. Prepare subs t rate-buffer mixture by mixing 5 volumes of phosphate buffer with 
one volume of olive oil emulsion by stirring. The pH of the buffered substrate should 
be close to 7.5. 

2. Measure 12 ml. portions of the substrate -buffer into test tubes (or flasks) 
using 2 tubes for each determination. Warm the tubes of substrate buffer mixture to 
37° in a water bath. 

3. Add 1.0 ml. serum to one of the tubes and mix thoroughly by gentle inversion. 
The second tube will serve as the blank. Incubate both tubes for 4 or for 16-24 hours 
at 37°C. 

4. At the end of the incubation period, add 1.0 ml. of serum to the blank. 

5. Immediately pour the contents of the tubes into a 100 ml. beaker and transfer 
completely by rinsing out the tube with 50 ml. of the 9:1 alcohol-ether mixture. Mix 
well with a glass stirring rod. 

6. Titrate the mixtures with 0.05 N NaOH to a "distinct" blue using 4-8 drops of 
thymolphthalein indicator (or titrate to pH 10.65 using a glass electrode pH meter.) 

Calculation : 

The final result Is expressed In terms of the volume of 0.05 N NaOH required to 
titrate the fatty acid liberated by 1 ml. of ppvum in 4 or in 16-24 hours. 

Example : A serum sample gave these results for the blank and test sample: 

Test sample 8. 20 ml. 0. 06 N NaOH 

Blank M 3.30 ml. 0.06 N NaOH 

4. 90 ml. difference 

Since . 06 N NaOH was used 

4.90 x {0.06/0.05) = 6. 88 units/ml. serum 

Interpretation: 

The determination of "lipase" is most often requested as an aid in the diagnosis 
of acute pancreatitis. The lipolytic enzyme found in serum of patients with acute pan- 
creatitis apparently is inactive when tested with substrates of low molecular weight 
such as tributyrin or ethyl butyrate, but active when tested with high molecular weight 
substrates such as olive oil or triolein emulsions. {See Esterase, p. 164.) 

Normally there are present in serum ; lipolytic enzymes (esterases) active wlien 
tested with low molecular weight substrates but little or none of the enzyme active 
when tested with high molecular weight substrates (lipases). 

The values found for "lipase" In cases of certain disease Btatcs are listed below: 
Condition 4 hour lipase 16-24 hr. lipase 

Normal 0.31 (0.06 - 0.89) (0.2 - 1.5) 

Acute pancreatitis 2-10 similarly elevated 

Note: 1. The incubation may be carried out in Erlenmeyer flasks to avoid subsequent 
transfers. 
2. The addition of alcohol-ether may be eliminated,if prcferred,lf good stirring 
and a slow approach to the end point is made. 

208 



( 



I 









MAGNESIUM (Titan Yellow) 
Serum 

References ; 

Nelll, D. W. and R.A. Neely; The estimation of magnesium in serum using titan 
yellow. J, Clin. Path., 9, 162 (1956). 

Heagy, F. C. ; Use of polyvinyl alcohol in the colorimetric determination of mag- 
nesium in plasma or serum by means of titan yellow. Can. J. Research, 26E . 
295 (1948). 

Princip le: 

The blood, serum, or plasma proteins are precipitated by meanB of tungstic acid. 
An aqueous solution of titan yellow is added to the Water-clear filtrate, and sodium 
hydroxide is added to develop the red magnesium hydroxide -titan yellow complex. Gum 
ghatti is used to stabilized the color lake. 

Reagents ; 

Gum ghatti 0. 1%. Powdered gum ghatti, 0. 1 g. , la suspended In a muslin bag In 
100 ml. of distilled water for 24 hours. (Polyvinyl alcohol 0. 1% can be used in place 
of gum ghatti. ) 

Titan yellow 0.05%. The dye powder, 0. 1 g. , la dissolved in 200 ml. distilled 
water. 

Stock Standard Magnesium Chloride. 8. 458 g. of MgCl * 6H O, Is dissolved in 
distilled water and made up to one liter. This solution contains 1,000 micrograms 
magnesium per ml. 

Working Standard Magnesium Chloride, One ml. stock standard solution is di- 
luted to 200 ml. with distilled water to give a concentration of 5 micrograms of magne- 
sium per ml. Volumes of 1, 2, 3, 4, and 5 ml. made up to 5 ml. with distilled water 
in each case and representing. 5, 10, 15, 20 and 25 micrograms of magnesium are 
used in setting up the standard curve. 

Calcium Chloride. 16. 1 3 mg. of CaCL' H„0 Is dissolved in distilled water and 
made up to 100 ml. , to give a final concentration of 0, 05 mg, Ca per ml. 

Sulfuric Acid 0. 67 N (reagent grade). 

Sodium hydroxide 4 N (reagent grade). 

Sodium tungBtate (Na 2 W0 4 * 21^0) 10%. 



209 



Procedure : 

1. One ml. of serum is diluted with 5 ml. of distilled water. 

2. Add 2 ml, of 10% sodium tungstate and 2 ml. of 0.67 N HqSO*. 

3. Mix and then centrifuge for 5 minutes at 2500 rpm. 

4. To 5 ml, of the protein-free supernatant Is added 1 ml. distilled water, 

1 ml. 0. 1% gum ghattl, 1 ml. 0.05% titan yellow solution and 2 ml. 4 N NaOH. 

5. A reagent blank using 1 ml. CaCl 2 solution containing 0.05 mg. Calcium In 
place of serum is treated similarly to the test. 

6. Compare photometrically at 540 mu. Set the photometer to read 100% trans - 
mlttance against water. 

Standard Curve : 

Headings of optical density are converted to magnesium concentrations by refer- 
ence to a standard curve. 

Prepare the standard curve by carrying out the above color reactions on 5 ml. 
samples of solutions containing 5, 10, 15, 20, and 25 micrograms of magnesium re- 
placing the 1 ml. of distilled water added to the protein-free supernatant in the test 
with 1 ml. CaCl 2 solution containing 0.05 mg. calcium. The standard blank is 5 ml. of 
distilled water. 

Calculations : 

., i O.D. T - O.D. B ■ „ „ 100 

Magnesium = *■ " x C g x iiii- 

O.D.q - O.D.x, *>.5 

o 

where: Cg - amount of Mg In standard in mg. 

O. D<r ; optical density of test solution 

O. D Q - optical density of standard 
o 

O.Dq - optical density of reagent blank 

0.% 0-600 

O.Dg 0.575 

O.D- 0.515 

Cg 0.01 

Then: 0-600 - 0-515 x 0.01 x 100. = 2.83 mg. Mg/l00 ^ 
0.675 - 0.515 0.5 

The value can also be obtained from the standard curve. 

Interpretation: 

Normally about 1 to 3 mg. of Mg are found in 100 ml. of serum and about 1.6 mg. 

per 100 ml. of blood. 

Decreased Mg levels have been observed In severe renal disease, toxemia of 
pregnancy and chronic alcoholism. Increased Mg levels have been observed in Mg 
poisoning from the Ingestion of epsom salt in severe renal disease. 

210 



Example . 






MAGNESIUM 
Serum 

References : 

1. Uriggs, A. P., J. Biol. Chem. 52, 349-355 (1922). 

2. Dennis, W. , J. Biol. Chem. 52, 411 (1922). 

3. Fiske, C.H. , J, Biol. Chem" 66, 375 (1925). 

Principle : 

Magnesium is determined In the serum, following removal of calcium. The magne- 
sium is precipitated as magnesium ammonium phosphate (MgNH 4 PO.'6H„0). This 
compound Is treated with ammonium molybdate to form phosphomolybdlc acid, which 
Is then reduced to molybdenum blue by semidlne or amino-naphthol-sulfonic acid. The 
molybdenum blue (uncertain composition) is then determined photometrically. 

Reagents : 

1. 2% (w/v) potassium dlhydrogen phosphate. Dissolve 2 g. KH„PO. (reagent 
grade) in 100 ml. water. Filter this solution each time just before use. 

2. Ammonium hydroxide. Concentrated reagent grade (sp.gr. 0.9). 

3. Alcoholic wash solution. Add 200 ml. of 95% (v/v) ethyl alcohol and 50 ml. con- 
centrated ammonium hydroxide to a 1000 ml. volumetric flask. Fill to the mark with 
water and mix thoroughly. Keep in refrigerator. 

4. Ammonium molybdate reagent. O.0O83_N. Place 2. 566 g. of reagent grade 
ammonium molybdate In a 250 ml. volumetric flask. Add slowly about 175 ml. of dis- 
tilled water and 35 ml. concentrated H„SO. (sp, gr. 1, 84) while swirling the flask. 
Then add water to the level of the neck of the flask and allow to cool. Now add water 
to the mark and mix. 

5. N-phenyl-p-phenylenediamine monohydrochlorlde (se ml dine -Eastman Kodak Co. 
#2043). Place 60 mg. of the salt In a 100 ml. volumetric flask. Wet the salt with a 
few drops of ethanol and fill the flask to the mark with 1% aqueous sodium bisulfite 
solution. Shake the flask vigorously while filling. Filter the resultant cloudy, purplish 
solution, and store the colorless, clear filtrate in a brown bottle in the refrigerator. 
Discard this reagent when the blank becomes excessively colored. 

6. Amino -nap hthol -sulfonic acid (may be used Instead of semidlne). Dissolve 30 g. 
of sodium blBulf ite (NaHSOg) and 1 g. of sodium sulfite (Na-SCy 7H-0) In 200 ml. water. 
Add 0.5 g. of l-amino-2-naphthol-4-sulfonlc acid and mix thoroughly. Place in a dark 
glass bottle. This colorless solution develops a yellowish discoloration after about two 
weeks. Thus, it must be discarded at that stage. 

7. Phophorus stock standard. Place 433. 1 mg. of potassium dlhydrogen phosphate 
Into a 100 ml, volumetric flask. Add water to the mark. Dilute 20 ml. of the stock 
standard to 1000 ml. with distilled water for use as a working standard. Each ml. of 
the working standard contains 0.02 mg. of phosphorus. 



211 



Procedure : 

1. Follow alepB 1-6 of the calcium procedure on page 119. Transfer the supernate 
obtained In Step 6 to a 12 ml. heavy duty centrifuge tube. 

2. Add 1 ml. of 2% KH PO . and 1 ml. of concentrated ammonium hydroxide to this 
centrifuge tube. 

3. Mix thoroughly and help initiate precipitation by rubbing the Inside of the tube 
with a glass rod. 

4. Rinse glass rod with several drops of water before removing. 

5. Stopper the tube and place In refrigerator for 2 hours. 

6. Centrifuge at 1500 rpm for 30 minutes. 

7. Decant supernate carefully and discard. 

8. Add 5 ml. of chilled alcoholic wash fluid. Tap tube until the precipitate Is re- 
suspended. 

9. Centrifuge at 1500 rpm for 30 minutes and again discard the supernate. 

10. Add 5 mi. water and tap the tube to resuspend the precipitate. 

11. Prepare 2 standards in similar tubes. Place 2 and 4 ml. of phosphate working 
standard In these tubes respectively. Dilute to 5 ml. (by adding 3 and 1ml. HLO re- 
spectively). 

12. To each tube add 0.5 ml. molybdate reagent. 

13. Prepare the blank by adding 5 ml. water and 0.5 mi. molybdate in a similar tube. 

14. To each tube add either 5 ml. eemidine reagent or 0. 4 ml. amino -naphthol- 
sulfonic acid reagent. Mix each tube Immediately after addition of this reagent. 

15. Let stand five minutes. Compare photometrically in a photometer at 660 mu. 

Calculations : 

1. In a molecule of MgNH.PO^, 31 mg. of phosphorus is equivalent to 24 mg. of 
magnesium. 

2. The working phosphate standard contains 0.02 mg. P/ml. 

°- 04m K- P x 24 m *' M K- x 1 me( i- M S- x 1000 = 1.29 meq. Mg/Uter 
2 ml. 31 mg. P 12 mg, Mg ^ 

a. Thus the 2 mi. standard represents 1.29 meq. Mg/liter. 

b. The 4 ml. standard represents 2.58 meq. Mg/liter. 

3. A calibration curve may be prepared by plotting optical density versus Mg in 
meq/liter on equal axis graph paper or the calculation may be made using the nearest 
standard. 

(D a /Dj x C x ^P- -_ Mg (meq. /liter) 

For 2 ml. standard z (D u /D s ) x 0.00258 x M9£ me q. /liter 

r (*>,./!> J " 1-29 meq. /liter 
u s 

For 4 mi. standard = (D„/nj x 2.58 meq. /liter 

Lt g 

212 



NITROGEN (TOTAL) 
Macro-Kjelduhl Method 



Reference: 



Hiller, A., Plazin, J. . andVanSlyke, D.D.: J. Biol. Chem. 176,1401 (1948) 



Principle 



By digestion with concentrated H,,S0 4 and a catalyst such aa copper sulphate, all 
of the nitrogen of the sample Is converted to (NH 4 ) 2 S0 4 . The ammonia is then liberated 
by making the solution strongly alkaline with NaOH. Distillation of the NH 3 Into boric 
acid solution is then carried out. The NH in the boric acid is then titrated with stand- 
ard acid. 

Reagents : 

1. Concentrated H„SO, 

2. 10%CuSO 4 .5H 

3. 40% NaOH. Dissolve 40 g. of NaOH in water. Cool. Dilute to about 100 ml. 

4. 0.1NHC1, standardized 

5. Boric Acid, 2% 

6. Mixed indicator, five parts of 0. 1% bromcresol green in alcohol plus one part 
of 0. 1% methyl red In alcohol 

7. Zinc metal, mossy 

Procedure : 

1. Into a 300 ml. Kjeldahl flask pipet exactly 5 ml. of urine or 1.0 ml. of serum 
(or other volumes of other materials; they should contain between 1.0 and 5.0 mEq. of 
nitrogen). Add accurately with a cylinder 10 ml. of cone. HgSO.. Add 2 ml. of 10% 
CuS0 4 -5H 2 solution. 

2. Digest on a digestion rack until the solution is clear and faintly blue-green in 
color. 

3. Cool. Add 150 ml. of HgO. Mix. Cool. 

4. Pour in, to run under the acid layer, 45 ml. of 40% NaOH solution. (DANGER 1 
Do not spill I ) 

The acid layer above the NaOH prevents loss of ammonia. Add a small piece of 
mossy zinc to promote smooth boiling. 

Without shaking connect the flask through a spray trap to a condenser. 

5. Into a 250 ml. Erlenmeyer flask place 50 ml. (measured with a cylinder) of 2% 
boric acid, and 0. 5 ml. of mixed indicator solution, place the flask so that the glass 
delivery tube at the end of the condenser dips just below the surface of the acid. Start 
water flowing around the condenser. 

6. Now shake the Kjeldahl flask to mix up the acid and alkaline solutions. A deep 
blue color should appear. Heat the Kjeldahl flask and distill 100 ml. of Uquid into the 
Erlenmeyer flask. 

7. When 100 ml. have distilled over, lower the Erlenmeyer flaBk until the delivery 
tube is out of the solution and distill a few ml. more to rinse out the inside of the tube, 

213 



Rinse outside of delivery tube with dhitilled water using a wash bottle. 

8, Titrate Uie ammonia wild 0. 1 NJICl to the loan of blue-green color and the ap- 
pearance of a "gray" or neutral color, Sec discussion below. Determine the "blank" 
correction as directed below and subtract it from the volume of acid used. 

Calculations : 

Take N as the normality of the standard acid, of which a total of A ml, was used, 
B as the blank correction, and V as the volume of 24-hour urine. Since a 1 molar solu- 
tion of NH„ contains 14 g. of N per liter, 

(A - B) x N x 0.014 x (V/5) = g. nitrogen per 24 hours 



Discussion: 

The color change of the indicator is indicated by the following data: Blue purple - 
pH 4. 5; Blue-green - pH 4. 9; pink - pH 4.3. The end point being between the pink 
and the blue - where the solution appears gray. 

To determine the blank, introduce into a thoroughly clean 250 ml. Erlenmeyer 
flask 50 ml. of 2% boric acid, and 0.5 ml. of mixed indicator solution. Add water to 
volume of the unknowns and titrate as above. This represents a titration (or indicator) 
blank. For a complete reagent blank a complete digestion and distillation should be 
carried out without adding a sample. ( 



214 



(_ 



NON-PROTEIN NITROGEN IN Dl.OOD 
Micro-Kjcldahl Method 

Reference : 

Folin, O. , and Wu, H. : J. Biol. Chem. 38, 81 (1919) 
Hoffman, W.S.: J. Lab.and Clin. Med., 25, 856 (1940) 

Principle : 

A protein-free filtrate is first prepared by the tungstic acid procedure. Then all 
organic compounds present in protein-free filtrate are destroyed by oxidation with a mix- 
ture of H 2 S0 4 and H„P0 4 . AH nitrogen present in these compounds Is converted to HH,. 
The solution is diluted and added to alkaline Nessler'B reagent, developing color in 
proportion to the amount of ammonium Ion. Photometric comparison is made with a 
solution containing a known amount of nitrogen in the form of ammonium Ion. 

Reagents : 

Use ammonia-free distilled water for all solutions and dilutions. 

1. Ammonium sulfate, stock solution. Dry some pure ammonium sulfate In a vacu- 
um desiccator over a drying agent for 24 hours. Weigh out 0. 9434 g. , dissolve in water, 
add 1 drop of cone, sulfuric acid, dilute to exactly 1 liter, and mix. 1 ml. contains 0.2 
mg. of nitrogen, 

j2. Ammonium sulfate solution, dilute standard. Dilute exactly 15 ml. of the stock 
solution up to 100 ml. in a volumetric flask and mix. 5 ml. contains 0. 15 mg. of nitrogen. 

3. Alkaline Nessler's reagent for acid digestion mixtures. Dilute 135 ml. of stock 
mercuric potassium iodide solution (see Urea Nitrogen, p. 2.65) plus 800 ml. of 2.5 N 
NaOH with water up to 1 liter and mix. This solution should be made several days ahead 
of use. Any Bediment that forms should be allowed to settle,, and only clear supernatant 
liquid should be used. The amount of alkali given here is adapted for the use of 15 ml. 

of the reagent in a total volume of 50 ml. of Nesslerized solution, when 20 milliequlva- 
lentR of an acid are to be neutralized in the digestion mixture. 

4. Acid digestion mixture for non-protein nitrogen. Dissolve 0.6 g. of CuSO.- 
5H z O in 100 ml. of water, add 75 ml. of cone. H 2 S0 4 and 25 ml. of 85% HjPO. and 
mix. 

5. 2% Gum ghattl solution. Tie 2 g. of gum ghattl tears In a bit of washed gauze 
and hang for 24 hours near the top of 100 ml. of water in a cylinder. Use a few drops 
of toluene as a preservative. Discard the undissolved portion, mix, and filter through 
cotton. Keep in the refrigerator. Use 1 drop (about 0.05 ml.) for each 10 ml. of final 
volume of Nesslerized solution. This acts as a protective colloid. 

Procedure : 

1. Into a 25 x 200 mm. pyrex test tube graduated at 35 ml. place 5 ml. of tungstic 
acid protein-free blood filtrate. Add 1 ml. of acid digestion mixture and a glass bead 
to prevent bumping. Using a microburner, boil off the water. Lower the flame as low 
as possible, and continue to heat until acid fumes (dense white) fill the tube. Then 
cover with a watch glass or a small funnel. See that the heat from the small flame does 
not strike the tube above the surface of the liquid. 

215 



2. The liquid turns dark and then grows tighter. As soon an all brown or yellow 
coior haa disappeared, and the color Is a faint Mw-greeh, cease heating. The heating 
period should bo as abort as possible. 

3. Allow to cool until the tube can he handled, 

4. Add about 5 ml. of water ami make sure all the syrupy liquid goes Into solution. 
Dilute to about 30 nil. and add 5 drops of 2% gum gUtttti solution. Cool In a water bath 
with the standard to about 20°C. Dilute to exactly 35 ml. In a clean, dry 100 ml. beaker 
place exactly 15 ml, of NcBslor's reagent (for acid digestion mixtures). Rotate Hie lat- 
ter and pour in rapidly the 35 nil. of unknown solution. Pour back and forth several 
times to mix. 

5. Prepare a standard solution. In a test tube graduated at 35 ml. , place 6 ml. of 
standard ammonium sulfate solution containing 0. 15 mg. of nitrogen. Add 1 ml. of 
above (no heating required). Nesslerke the standard, the unknown, and the blank simu- 
taneously and read in the photometer (515 mu) after 10 to 20 minutes. The blank con- 
taining digestion mixture and gum ghatti, Is treated in the same way as the standard, 

Calculations: 



5 ml. of tungstic acid filtrate corresponds to 0.5 ml. of blood. Then the non-protein 
nitrogen (NPN) found is: 

<D U /D fl ) x 0.15 x (100/0.5) = (D U A> B > x 30 (mg. N.P.N./100 ml. blood) 

Comments : 

If a concentrated solution of phoophoric acid is heated too long or at too high a tem- 
perature, it attacks glass. The resulting silicious material may cause turbidity at a 
later step. Such turbidity most often appears when there has been carelessness or lack 
of skill in the heating. 

The process of Kesolerization Is U3ed frequently in biochemical work. After mixing 
the color Increasas gradually. Color is due to a colloidal solution of NH„Hg_I (formula 
in doubt). See Nichols and VVillits: J. Am, Chem. Soc. J56, 7G9-774 (1934). TWb com- 
plex salt is very insoluble and can be used for gravimetric determination of ammonia in 
large amounts. However, it can bs made, by proper selection of conditions, to form a 
clear colloidal solution well adapted to photometric determination. Once the Neosler- 
I'.cd solution becomes turbid the analysis is ruined. The necessary conditions for se- 
curing clear solutions are: 

1. The concentration of ammonia (NH^) should not be too high {leas than 1 mg. per 
ml. of final solution). 

2. Proper alkalinity.. Free OH~ ions should be present at a concentration of 20 MEq 
per 100 ml. of final solution. 

3. Temperature of the solution should be low — 20°C. or lower. 

4. Mixing of the alkaline Nessler reagent with the ammonia solution must be instan- 
taneous. Otherwise for an appreciable length of time, an unsuitable concentration of OH" 
ions will exist, and the precipitation of the colloid may begin. 

5. The presence of a protective colloid (gum ghatti) greatly facilitates the mainten- 
ance of the colloidal state. 

6. The concentration of neutral salts should be low. 

7. See also p. 2G6. 

210 






4. Sodium hydroxide 0, 5 N. Standardize and adjust to between 0. 49 and 0. 51 N. 

5. Sodium bicarbonate 0. 5 M. 

Dissolve l. 2 g. NaliCO., In distilled water and dilute up to 100 ml. 

6. 4-amlno-antipyrine 0.6% (w/v). 

Dissolve 0,Gg. of 4-amino-antipyrine in distilled water and make up to 100 ml. 

7. Potassium ferricyanidc 2. 4%. 

Dissolve 2. 4 g. K 3 Fe(CN) 6 In distilled water and dilute up to 100 ml. 

8. Stock phenol standard 

Dissolve 1, 2 g. pure crystalline phenol and dilute up to 1 liter using about 0. 1 N 
IIC1 as the solvent (8.5 ml. concentrated HCl diluted up to 1 liter). Standardize as out- 
lined below. 

9 k Dilute phenol standards 

(a) Weak working standard. 

Dilute 1.00 ml. stock phenol standard up to 100 ml. with distilled water, Add 
2-3 drops of toluene. (1 ml. = 0.01 mg.) 

(b) St r on g wo r king s tan da r d . 

Dilute 3,00 ml. stock phenol standard up to 100 ml. with distilled water. Add 
2-3 drops of toluene. (1ml. = 0.03 mg.) 
Store under refrigeration. Good for one month. 
10. Tartrate buffer 0. 2 M pH 4.9 

Dissolve 3.00 g. L(/) tartartlc acid-analytical grade (dextro-rotatory tartaric 
acid) In 50 ml. distilled water. Add about 35 ml. 1 N NaOH, and adjust the pH to 4.9. 
Dilute to 100 ml. Add two drop* of toluene and store under refrigeration. 



Procedure : 

Into a large test tube whose contents can be easily mixed by shaking carry out the 
additions and operations listed below in the tables. 



A. Control and Test 



Control (ml.) 



Test (ml.) 



Carbonate Buffer 
Substrate 



1.0 
1.0 



1.0 
1.0 



Mix and warm 3 minutes at 37°C. in a water bath. 



Plasma 



0.1 



Mix and warm 16 minutes at 37°C. in a water bath. 



NaOH 0.5 N 
Plasma 
NaHCO 3 0.5 M 
A.A.P. 0.6% 
K 3 Fe(CN) fi 2.4% 



0.8 


mix 


0.8 


mix 


0.1 


mix 







1.2 


mix 


1.2 


mix 


1.0 


mix 


1.0 


mix 


1.0 


mix 


1.0 


mix 



219 



B. Blank and Standards 



Carbonate buffer 
Phenol standard 


Blank 
1.1 


Standard 

(0.01) 

1.1 


Standard 
(0.03) 
1.1 


0.01 
0.03 
Water 


1.0 


1.0 


— 
1.0 


NaOHO.SN 
NaHCO 3 0.5 M 
A. A. P. 0.6% 
KgFe(CN) 6 2.4% 


0. 8 mix 
1. 2 mix 
1.0 mix 
1.0 mix 


0.8 mix 
1.2 mix 
1.0 mix 
1.0 mix 


0.8 mix 
1.2 mix 
1.0 mix 
1.0 mix 


Acid Phosphatase 








A. Control and Test 


Control 


Test 





Citrate buffer 
Substrate 

Plasma 

NaOHO.SN 
Plasma 

NaHCO 3 0.5M 
A. A. P. 0.6% 
K 3 Fe(CN) 6 2.4% 



1.0 




1.0 


1.0 




1.0 


Mix and 


warm 


-3 minutes 37°C. 







0.1 ml. 


Mix and 


warm 


- 1 hour 37°C. 


1.0 mix 




1.0 mix 


0.1ml. 




— _ 


1.0 mix 




1.0 mix 


1.0 mix 




1.0 mix 


1.0 mix 




1.0 mix 



B. Blank and Standards 

Citrate buffer 
Phenol standard 

0.01 

0.03 
Water 

NaOHO.SN 
NaHCO„0.5M 
A. A. P. 0.6% 
K 3 Fe(CN) 6 2. 4% 



Blank 
1.1 

1.0 

1.0 mix 
1.0 mix 
1.0 mix 
1.0 mix 



St andard] 
1.1 
1.0 



1.0 mix 
1,0 mix 
1.0 mix 
1.0 mix 



Standard^ 
1.1 

1.0 

1.0 mix 
1.0 mix 
1.0 mix 
1.0 mix 



Allow the color to develop for 15 minutes and read against a distilled water 
optical blank at 510 mu. B the transmittance of any of the unknowns is between 6 and 
10%, th= analysis may be saved by diluting control and test solutions, and blank and 
standard solutions, with an equal volume of water. Mix and compare photometrically, 
as before. The color may fade rapidly after 60 to 120 minutes due to (a) phenol or 
(b) reducing substances. 

220 



( 



(. 



D t. 


" D c 


D a 


-Db 


D t - 


-De 



Calculation : 

A King-Armstrong unit of alkallnp phosphatase is defined as the amount of enzyme 
which will liberate one mg. of phenol from a 0. 005 M oolutlon or dlnodlum phenyl phos- 
phate in 15 minutes at 37°C. In bicarbonate buffer lit pH 10. 

A King-Armstrong unit of acid phosphatase 1b defined as the amount of enzyme 
which will liberate one mg. of phenol from a 0. 005 M solution of .dlsodium phenyl phos- 
phatase in one hour at 37°C. In citrate buffer at pH 4, 9. 

Phosphatase units - — L- — &■ x 0.01 (or 0.03) x Hi 

0.1 

Dg _ ^ x 10 (or 30, 

where D refers to optical densities of 

t = test; c - control; s - standard and b « blank 

Prostatic Acid Phosphatase (tartrate sensitive fraction) 

Proceed as under acid phosphatase but use a mixture of 8 parts citrate buXfer 
pH 4. 9 and 2 parts . 2 M tartrate buffer pH 4. 9. It is not necessary to use tartrate 
buffer for the blank, standards or the control tube since the color production is not 
affected by tartrate. 

Interpretation : 

Alkaline Phosphatase . 

Normal values - adults 4-10 King-Armstrong units 

-children 10 - 20 King -Armstrong units 

Elevated values are seen In disorders of bone metabolism and in hepatic and bil- 
iary tract disease; for example, rickets, hyperparathyroidism, hepatocellular damage, 
and biliary obstruction. 

Acid Phosphatase 

Normal values 0-4 King-Armstrong unite 

Increased values are seen In prostatic carcinoma with metastases. Levels which 
are inhibited to an extent of more than one King -Arm strong unit by tartrate are said to 
be indicative of prostatic malignancy. 

Standardization of Phenol 

Reagents: 

1. Phenol stock standard - about 1 mg. per ml. 



221 



( 

2. Sodium thlonulphatc (Na«B«0^ 0, 1 N. 

Prepare as outlined imder Secondary Standards, p. 29. 

3. Iodlne-potasBium Iodide Bolutlon 0.1 N. Dissolve 25 g. potaBBium iodide 
(KI) in 25 ml. of distilled water, add 12.7 g. resublimed (analytical grade) I 2 , dissolve 
and dilute up to 1 liter. 

4. Potassium bi-iodate KH(IOg) 2 0.1N (or Potassium lodate KIO3 0.1 N.) 
Prepare as outlined under Primary Standards, p. 23. 



Procedur e: See p. 29, Iodlmetry. 






A. Standardization of 0. IN sodium thlosulfate 

1. Into a 250 ml. Erlenmeyer flask pipet 5 ml. of 6 N hydrochloric acid, add 2 g. 
potassium iodide and 3-5 ml. of water. 

2. Transfer 25, 00 ml. of standard bl -lodate (or iodate) to the flask. Tree 
iodine will now appear in the solution. 

3. Titrate the liberated iodine with 0.1N sodium thlosulfate and calculate the 
thlosulfate concentration. (See p, 18 Volumetric calculations.) 

B. Standardization of Iodine -potassium iodide 0. 1 N Bolutlon. 

1. 25.00 ml. of ^'KI solution are transferred to a 250 ml. Erlenmeyer flask 
and titrated with the standardized sodium thlosulfate solution. 

2. Calculate the Iodine concentration as above (A3), 

C. Standardization of stock phenol standard. ( 

1. Transfer 25.00 ml. of stock phenol solution to a 250 ml. glass-stoppered 
Erlenmeyer flask. Add 50 ml. 0. 1 N NaOH and heat to 65°C. (not higher). Add 25. 00 
ml. of the previously standardized iodine -potassium iodide solution and mix. 

2. Stopper the flask and allow to stand at room temperature for 45 minutes. 

3. Add 5 ml. of concentrated hydrochloric acid and titrate the excels iodine 
with the previously standardized sodium thlosulfate solution. 

Calculation : 

Mg. phenol per 25 ml. « 1.568 x (ml. 0.1 Nig -ml. 0. 1 N thlosulfate.) Adjust 
the concentration of phenol to exactly 1.00 mg. per ml. and store under refrigeration. 



Example: 

A. Standardization of 0. 1 N sodium thlosulfate 23.45 ml. of sodium thlosulfate 
were required to titrate 25. 00 ml. 0. 1 N KH(I0 3 ) a 

25 - 00 x 0.1 = 0.1065 N sodium thlosulfate 
2345 Factor Is 1. 065 

B. Standardization of 1 9 • KI solution. 24. 87 ml. of 0. 1065 N sodium thlosulfate 



222 



were required to titrate 26.00 ml. of I„ * KI solution. 



24.87 x 0.1065 
25.00 



0.1060 N 13- KI solution 
Factor is 1.060 



C. Standardization of stock phenol standard solution. 6. 90 ml. of 0. 1065 N sodium 
thiosulfate were required to titrate the iodine excess. 



Mg. phenol per 25 ml. = 1.668 (25.00 x 0.1060 - 6. B0 x 0.1065) 
- 1.568 (26.50 -7.35) 
* 1.668 x 19.15 - 30.00 mg. phenol 
D. Dilution Calculation, 

30.00 _ 1.2 mg. phenol per ml. 









25.00 

If 970 ml. of the solution remain: 

970 x 1.2 a X x 1.0 
970 x 1.2 






X = 



1.0 



- 1163 ml. 






1163 - 970 = 193 ml. 0. 1 N HC1 to be added to give 1. 00 mg. phenol per ml. 


















223 



phosphate, mona\Nic 

Scrum 

References : 

1. Fiske, CH. and Subbarow, Y. , J. Blol.Chem. 66, 375 (1925) 

2. Gomori, G, , J. Lab. & Clin. Med. 27, 955 (1941-42) 

3. Dryer, R.L., Tammes, A.R., and-Routh, J.I, , J. Biol. Chem. 222, 177 (1967) 

Principle : 

The inorganic phosphorus present In a trichloroacetic acid protein-free filtrate 
of serum in the form of the orthophosphate (salts of orthophosphoric acid, H,POj) 
react with molybdate In acid solution, and a blue color Is formed from the resultant 
"phosphomolybdate" after the addition of a reducing agent, iuch as "plctol" or "metol" 
which is p-methylamlno-phenol (sulfate). 

Reagents : 

1. Trichloroacetic acid 5% w/v in water. ) 

Trichloroacetic acid 10% w/v In water.) Keep refrigerated 1 Important! 

2. Sulfuric acid 10 N. Carefully pour 100 ml. of concentrated H-SO. Into 200 ml. 
of water, in a mixing cylinder (Pyrex), mix. After cooling dilute to 360 ml. (exactly) 
with water. 

3. Molybdate II for serum (2.5% ammonium molybdate in 3. N H 2 S0 4 ). In a 1 
liter mixing cylinder dissolve 25 g. of the salt in 300 ml. of 10 N H 2 S0 4 and dilute 
to a liter with water. 

4. "Plctol" stock. Dissolve 3 g. NaHSOg In 100 ml. H 2 and add 1 g. "plctol " 
(Mallinckrodt). Shake. Store in brown bottle. Refrigerate. It will keep Indefinitely. 

6. Dilute "plctol." To 10 ml. "plctol" stock add 90 ml. HgO. Mix well. Prepare 
on day of use . 

6. Stock standard phosphate. KH 2 PO. is well dried. 0.3514 g. transferred to a 1 
liter flask / 10 ml. 10 N H 2 S0 4 , dilute to mark (6 ml. = 0.4 mg..P). 

7. Dilute standards: Prepare 2 standard solutions weekly as follows from stock 
phosphate . 

a. Strong working standard: 

Transfer 5 ml. stock std, phosphate to 100 ml s vol. flask, dilute to mark 
with 5% trichloroacetic acid (5 ml. = 0.02 mgm.). 

b. Weak working standard: 

Same except use 2 ml. (5 ml. s 0.008 mgm,). 

Procedure: 

1. To a glass-stoppered test tube or a glass -stoppered bottle, add exactly 9.0 ml. 
of 10% trichloroacetic acid. 

2. Add exactly 1. ml. of serum, mix well. Allow to stand 6-10 minutes and cen- 
trifuge until clear or filter through a #40 Whatman filter paper. 

3. To a cuvet or colorimeter tube add exactly 5.0 ml. of filtrate. 



224 



( 



( 



4. Prepare a blank by pipetting 5.0 ail. of 3% triehloruaeetic Into a cuvet. 

5. Prepare two standards In a similar maimer. 

a. 5 ml, a 0.02 nig, 

b. 5 ml. = 0.008 mg. 

6. Add 1.0 nil. molybdate II to each cuvet and mix. 

7. Add 4. ml. dilute pictol to each cuvet, mixing Immediately after the addition 
to each cuvet in succession. 

8. Read in a photometer after 10 minutes and before 60 minutes at 630 mu (or 
using a red filter) against the reagent blank set at 100. 

Calculation : 

Mg. P/100 ml. serum « (D u /D 3 ) x C 3 x 2M 

0. 5 

for strong standard; (D u /D 8 ) x 0.02 x I°-£ • (D u /D fl ) x 4 « Dog. pst cent 

0.8 

for weak standard: (D u /D 8 ) x 0.008 x 1M s (D u /D s ) x 1.6 - mg. per cent 

Urine : 

1. Dilute exactly 1. ml. of urine to 50 ml. with 8% trichloroacetic acid in a 50 ml. 
volumetric flask, mix well. Filter if not perfectly clear through Whatman #40 filter 
paper. 

2. To a cuvet add 5. ml. diluted urine. 

3. Add 1.0 ml. molybdate II. 

4; Add 5.0 ml. dilute pictol and mix Immediately. 

5. Compare in a photometer with blank and standard nrepared as above for serum. 

Phosphatase , Alkaline and Add; 

Reference : 

Shinowara, G. Y. , Jones, L. M. , and Reinhard; The Estimation of Serum Inor- 
ganic Phosphorus and Acid and Alkaline Phosphatase Activities, J, Biol. Chem. 142 , 
921 (1942). 

Principle : 

The alkaline and acid phosphatase activity of serum is estimated by allowing serum 
to Incubate at 37°C. with an organic phosphate ester, (B-glycarophosphate) at an appro- 
priate pH, pH 8. 7 for alkaline and 5.0 for acid phosphatase. The liberated inorganic 
phosphorus is then determined 

The Bodansky phosphatase unit is defined as that amount of phosphatase which will 
liberate one mg. of inorganic P from the substrate In one hour. 



225 



Vpparatua : 

1. An far phosphate. 

2. Glass-[3topptircd bottlce. 

3. Incubator net at 37°C. 

Reagents : 

1. Aa for inorganic phoBphorus. (see above). 

2. Buffered substrate-stock solution. A mixture of: 

Sodium D -glycerophosphate 2.50 g. and 

Sodium barbiturate (sodium barbital USP) 2. 12 g, ia diluted up to 250 ml. 
Mix. Preserve stock substrate in refrigerator In small glass -stoppered 
bottles.{125 ml.) under 3 ml. redistilled petroleum ether (BP 30-36°C.) 
to delay absorption of C0 2 from the air. 

3. For alkaline phosphatase: To 25 ml. alkaline buffered substrate stock add 
25 ml. distilled water. Mala; up dally as needed. 

4. For acid phosphatase: 25 ml. buffered substrate stock Is added to 25 ml, 
0. 1 N CH„COOH. Mix well. Do not attempt to store. 

0. 1 N CH 3 COOH. Weigh out enough glacial acetic acid to give 6.00 gm. of 
acid. Dissolve and dilute to 1000 ml. Mix well. 

Method : 

Alkaline Phosphatase: 

1. Prepare a reagent blank and two standards as outlined in Steps 4 and 5 In 
Inorganic Phosphorus p. 225. 

Procedure for control sample: 

1. Plpet 5.0 ml. dilute alkaline substrate Into glass-stoppered bottle. 

2. Add 4.5 ml. 10% CClgCOOH. 

3. Add 0.5 ml. serum, stopper. Mix and filter. 

4. Plpet 5 ml. of the filtrate Into a cuvet. 

Procedure for enzyme sample : 

1. Plpet 5.0 ml. dilute substrate Into glass-stoppered bottle. 

2. Place In Incubator to reach 37° C. 

3. Add exactly 0.5 ml. serum. Mix, note time. 

4. Remove at end of one hour. 

6. Immediately add 4. 5 ml. 10% CClgCOOH. 

6. Mix and filter. 

7. Plpet §,Q ml. of the filtrate Into a cuvet. 

Develo pment of color : 

Add 1.0 ml. of molybdate H, mix. Add 4.0 ml. of dilute plctol, mixing each cuvet 

immediately after the addition. 



220 






Photo metry: 

Set blank at 100 :md read control sample and enzyme sample and standards, after 
10 minutes and before 60 minutes using G80 mu or a red filter. 

Calculation : 

(D u /D 8 ) C g — — D nig. P/100 ml. serum (calculated as serum phosphorus), 

One phosphatase unit will liberate one tng. of inorganic phosphate from 

glycerophosphate In one hour under the specified conditions. 
P e a mg. P/100 ml. serum in Incubated sample. 
P e mg. P/100 ml. serum in unincubated sample (control). 
P e - P c = phosphatase units/100 ml. serum 

Example: 

P e = 8.0mg. % 
P c = 3.3 mg. % 
Phosphatase =.4.7 Bodansky unlts/100 ml. serum. (8.0 - 3.3). 

Precaution: 

1. As for inorganic phosphate. 

2. Exact time and temperature control Ib important. 

Acid Phosphatase: 

: *: — , 

Procedure : As in alkaline phosphatase except using the acid substrate. Use female 
serum control for check of the method. 

Interpretation : 

Phosphorus: Normal values — 5 mg.% in infants and children 

3.7 mg,% in adults 
In severe nephritis P may increase to 15-20 mg.%. In rickets P may fall to 
2 mg. % or lower. Insulin injection decreases P in serum due to formation of hexose 
phosphates. 

Phosphatase: 
Bodansky units 

Alkaline: Normal valueB 5-12 units In children 

1. 5-4.0 units in adults 

Increased in Paget's disease (up to 50x) 
Rickets (up to 20x) 

Hyperparathyroidism (up to 10X) 
Infectious Hepatitis (2-4X) 
Other hepatic diseases (2-10x) 
Acid: Normal values 0.0 - 1. 1 units 

Elevated only in 15-50% of cases of prostatic carcinoma with metastases 
(up to 30 units). 
Erythrocytes contain an acid phosphatase so hemolysis should be avoided. 

227 






PHOSPHORUS - Lipid 
So rum 






PrliuM nh); 

Total lipid;; r.re extracted :ta In the choleutcrol procedure. The alcohol-ether ex- 
tract la dried, r.nd dijp&tcd with sulfuric acid and hydrogen peroxide. The color is 
developed as uncbr tlie Inorganic phosphorus procedure. 

Procedure : 

1. Prepare an alcohol -ether extract of serum as outlined under Cholesterol, p. 152. 

2. Plpet 10.0 ml. of extract Into a 25 x 200 mm, test tube graduated at 25.0 ml. 

3. Evaporate to dryness using a hot water bath, avoid the use of an open flame. 

4. Add 2.5 ml. 5 J£ ^SO^ aa ^ a small quartz chip and digest until the water 
evaporates and the mixture turns brown or black. 

5. Allow to cool and add 1 drop of 30% H 2 2 directly into the mixture. 

6. Continue heating. Repeat HO treatment and reheating until colorless. 

7. Cool the tube, add a few ml. of water, heat to boiling. Cool Immediately, 
dilute the contents to 25.0 ml. Mix well. 

8. Transfer 5.00 ml. to a photometer cuvet. 

9. Proceed as in Inorganic Phosphorus, p. 224. 









Calculations : 

Since 0.4 ml. of eerum is represented by 10.0 ml. of filtrate 

C u = <D U /D 8 > C B X -— 

m (D u /D 8 ) C a x 250 * mg. lipid P/100 ml. eerum 






( 



( 





















228 






PORPHOBILINOGEN - Qualitative 
Urine 

liufenini '.e : 

WatK on, C. J. and 8. Schwartz. A simple teal for urinary porphobilinogen. 
Proc. Soc. Exp. Biol. L Med. 47, 393-4 (1941) 

Principle: 

Porphobilinogen in the urine forms a red colored compound with Ehrlich's Reagent 
in acid solution. This colored compound Ib Insoluble In chloroform. 

Reagents ; 

1. Saturated sodium acetate solution. 

2. Ehrlich's Reagent. (Add 0.7 grams of p-Dlraethylamlnobenzyaldehyde to 150 
ml. of concentrated HC1 and 100 ml, of water.) 

3. Chloroform. 

Procedure : 

1. To 2 ml. of urine In a test tube add 2 ml. of Ehrlich's reagent. 

2. Invert until mixed. Allow to stand 5 minutes. 

3. Add 4 ml, of saturated sodium acetate to the tube and invert until mixed. 

4. Note the color, If any. 

5. Add 1 ml. of chloroform and shake vigorously . Allow to stand until separated. 

6. Note the color of the chloroform layer. 

7. If a pink to red color develops In step four, porphobilinogen and/or urobilinogen 
may be present. If in step six the chloroform layer 1b red, while the urine layer be- 
comes colorless or tue original color of the urine, then porphobilinogen Is absent and 
urobilinogen is present. If the chloroform layer remains colorless and the urine layer 
is pink to red, then urobilinogen Is absent and porphobilinogen Is present. If both layers 
arc red, then both porphobilinogen and urobilinogen is persent. If large amounts of 
urobilinogen are present, the chloroform extraction may have to be repeated several 
times to remove all of the urobilinogen red, If both layers are colorless, then both 
porphobilinogen and urobilinogen are absent. 

8. Report porphobilinogen and urobilinogen as negative or positive. 

Notes ; 

Normal red-brown urines tend to disguise small amounts of pink color in the 
urine layer. 

I nterpretation : 

In acute porphyria the urine contains porphobilinogen which gives a positive 
Ehrllch reaction. Positive results may also be obtained in cases of liver disease. 
Urobilinogen is normally found In the urine as it partially escapes absorption by the 
liver and is excreted by the kidneys. 



229 



PORPHYRIN DETERMINATION 
Qualitative and Quantitative 

References ; 

Askevold, R. : Routine analysis of porphyrins in urine. Scandinavian J. of Clin, 
and Lab. Invest. 3. 318-9 (1951). 

Brunsting, L.K. , H.L. Mason and R. A. Aldrlch; Adult form of chronic porphyria 
with cutaneous manifestations. JAMA 146, 1207-12 (1951). 

West, E.S, andW.R. Todd: Textbook of Biochemistry, 2nd ed. (MacMillan Co. , 
New York) 485-7 (1955). 

Gradwohl, R.B.H. : Clinical Laboratory Methods and Diagnosis, 5th ed. 
(C.V. Mesh Co., St. Louis) 551-3 (1956). 

There are present in the excreta of the human body, pigments which have been Iden- 
tified as porphyrins. These pigments are closely related to the protoporphyrin found In 
the hemoglobin molecule, but are chemically distinct from it. In many diseases abnor- 
mal amounts of these pigments are excreted in the urine and feces. Therefore, a de- 
termination of the type and quantity of porphyrin present in the patient .can be a valuable 
diagnostic tool. 

A large variety of porphyrins are known to exist, some occuring naturally, while 
others have been synthesized. Of these the coproporphyrins and the uroporphyrins hav# 
been found to be particularly clinically significant with respect to certain diseases. A 
further specificity is observed in the isomers of both of these compounds; i.e. , copro- 
porphyria I, coproporphyria HI, uroporphyrin I and uroporphyrin 131 have been found to 
be particularly related to a number of specific diseases. 

A qualitative method for determining the presence of porphyrin, and a quantitative 
method for determining the amount of porphyrin present are here described. 

Qualitative Porphyrin Determination 

Prlnc iple : 

In many cases a qualitative test is sufficient to Indicate the presence of an abnorm- 
al quantity of porphyrins in the urine since the minute amounts that are normally pres- 
ent will not react to the test. Once the presence of the porphyrins has been established 
a quantitative test Is run to determine the degree of abnormality. 

The qualitative determination of the presence of porphyrins is based on a primary 
fluorescence In the near ultraviolet range of the spectrum exhibited by porphyrins In 
solution. However, since the fluorescence is easily masked by urinary impurities, 
the porphyrins must be separated from the original sample. This is accomplished by 
extraction, with a normal butyl alcohol -ethyl acetate mixture, from an acid aqueous 
solution. 



230 



( 






Should the test be positive, a further extraction with ether will separate the uro- 
porphyrins and the coproporphyrlns, permitting a more specific identification. Both 
exhibit a characteristic fluorescence in the near ultraviolet range. 

Apparatus : 

1. An ultraviolet light emitting In the near ultraviolet range of the spectrum. 

2. 50 ml. or 100 ml. separatory funnels. 

Reagents j 

1. Glacial acetic acid, 

2. 1:1 (v/v) mixture of n-butyl alcohol and ethyl acetate. 

3. 10% NaOH. 

4. Concentrated HC1. 

5. Ethyl ether. 

Procedure : 

1. Acidify a 15 ml. fresh urine sample with 3 ml. glacial acetic acid. 

2. Shake the acidified urine in a separatory funnel with an equal volume of the n- 
butyl alcohol-ethyl acetate mixture. Discard the aqueous phase. 

3. Wash the organic phase three times with distilled water. 

4. The organic phase will exhibit a red fluorescence under the ultraviolet light if 
porphyrins are present. If the red fluorescence is evident at this point proceed with 
the rest of the determination to determine which of the porphyrins are present. 

5. Extract the porphyrins from the organic phase with three portions (3 to 5 ml.) 
of 10% NaOH solution. The porphyrins go into the NaOH solution almost quantitatively. 
Discard the organic phase. 

8. Neutralize the NaOH solution with concentrated HC1 until the solution turns 
Congo Red paper gray. 

7. Add 1 to 2 ml. glacial acetic acid. 

8. Shake the solution with an equal volume of ether, in a separatory funnel. Copro- 
porphyrlns will transfer to the ether phase while the uroporphyrins will remain in the 
aqueous phase. 

If large amounts of porphyrins appear to be present, the aqueous phase may be 
extracted several times with ether to Insure the removal of the coproporphyrlns. 

9. Acidify the aqueous phase with HC1 and examine for red fluorescence under light 
in the near ultraviolet range. 

10, Extract the coproporphyrlns from the ether phase with HCl. Examine the aque- 
ous phase under the ultraviolet light. If coproporphyrlns are present a red fluorescence 
will appear. 

11. The coproporphyrlns and uroporphyrins are reported as positive or negative. 

Note: The qualitative procedure is summarized in Table I. 



231 



Table I 
Qualitative Determination of Porphyrins 






( 



15 ml. urine plus 3 ml. glacial acetic acid; add n-butyl alcohol-ethyl acetate mixture; 
separate organic and aqueous phases. 



Aqueous phase i Organic phase 

1 Wash with distilled water, three times 
Discard 



Wash water 



1 Discard 

•' 
i 

i 
i 

i 
i 
i 
i 

« 

i 
i 
i 
i 
i 
t 
i 
i 
i 



Washed organic phase. Extract with 
10% NaOH 



Extracted 


Alkaline aqueous phase. Neutralize 


organic 


with HC1; add 1-2 ml. glacial HAc; 


phase 


extract with ether. 


Discard 






Aqueous phase ' Organic phase 




Acidify with • Extract with HCU 




HC1. Fluor- J discard organic 




escence indl- , phase; red fluor- 




cates uropor- ' escence In aqueous 




phyrtns ! phase Indicates 




i coproporphyria. 






232 



Quantitative Porphyrin Determination 

Principle : 

Once the presence of the porphyrins has been established, the amounts present 
most be determined. This is done spectrophotometricallj for both the coproporphyria 
and the uroporphyrins, following isolation from sources of Interference In the original 
sample, and from each other. The coproporphyria (I and II Q are separated from the 
urine sample by extraction from the aqueous media with ether containing acetic acid. 
Uroporphyrin m is extracted from the aqueous media, at a pH of 3. 0-3. 2, with ethyl 
acetate. (Authentic uroporphyrin I is not extractable by ethyl acetate.) 

The maximum light absorption of the coproporphy rlns has been found to occur at a 
wave length of 401 mu while that of the uroporphyrins has been established at 406 ma. 
Thus a determination of the optical density of a solution containing the isolated porphy- 
ria will permit calculation of the porphyrin concentration in the original sample. 



1. Any spectrophotometer reading In the 380-430 mu range is satisfactory, although 
the Beckman DD la recommended. 

2. Separatory funnels. 



Heagente: 




1. 


Glacial acetic acid 




2. 


1.0 H acetic acid 




3. 


l.ON sodium acetate 




4. 


Ethyl ether 




S. 


3% (wt. /vol.) CaCl 2 solution 


6. 


5% (wt./vol.) Na 2 HP0 4 


solution 


7. 


l.ONNaOH 




8. 


O.lNNaOH 




9. 


0.5 N HC1 




10. 


0. 1 N HC1 




Coproporphy rin Procedure: 





1. Acidify 15 ml. of urine with 1 ml. of glacial acetic acid. 

2. Extract the acidified sample in a separatory funnel, with 30.0 ml. of ethyl 
ether. Discard the aqueous layer. 

3. Wash the ether layer, till clear, with distilled water. 

4. Extract the coproporphy rlns from the ether with five 1 ml. portions of 0. 1JN 
BC1. 

5. The total volume of acid extract will be very close to 5 ml. 

6. On the spectrophotometer, determine the optical density using 1.0 cm. cuvets 
of the solution at 380, 401 and 430 mu against a 0. 1 N HC1 blank. 



233 



r :°V '^porph yrin C al culations: 

Krom the determined optical densities the total micrograms In the sample, and the 
microgram per cent are determined. In equation (1) D corr (optical density corrected 
for nbsorptlou due to Impurities in the porphyrin solutions) ts calculated. 

(1) 2D 401 - ^380 J D 430 > d 

1.833 ' corr 

where D ~ optical density. I> corr = 0.65 correspondF (o 1 microgram of copropor- 
phyria per ml. Therefore, 

corr 

( 2 ) „ gc = micrograms coproporphyrin/ml. acid extract 
U. 65 

Then, the total micrograms of coproporphyria present In the acid extract is 

D 
coir 
@) n — x m ^- ac ^ extract a micrograms coproporphyrin 
0. 65 

This Is equivalent to the total micrograms of coproporphyrin present in the original 
urine sample. The microgram per cent is then determined by dividing the total micro- 
grams present by the original sample Bize, and multiplying by 100. 



IVnrr m ^- acid extract 
(4) — -■ ■ ■ ■■ x x 100 = microgram per cent coproporphyrin 

0. 65 ml. samnle 



Since, under normal conditions, the ml. acid extract - 5 and the ml. sample a 15, the 
formula may be simplified to 

corr 

(5) x 33.3 = microgram per cent coproporphyria 

0. 65 

The total urine volume, total micrograms coproporphyrin and the total microgram 
per cent coproporphyrin are reported. 

Uroporphyrin Procedure : 

1. Bring a 5 ml. urine sample to a pH of 5.0 to 5.5 with 5 ml. of acetate buffer. 
(Check with pH paper.) 

2. Place 2 ml. of the mixture in a centrifuge tube: add 2 drops of phosphate solu- 
tion, 2 ml. CaCU Bolution and 2 ml. 1. N NaOH. Mix well. 

3. Centrifuge at 1500 rpm for 10 minutes. Discard the supernatant liquid. 

4. Wash the precipitate with 0.1 N NaOH, centrifuge, and decant supernatant liquid. 
6. Wash twice with distilled water, centrifuging and discarding supernatant liquid 

each time. A red fluorescence (indicating porphyrins) under an ultraviolet light should 
not be present at this stage. If this occurs the unprecipitated porphyrins may be pre- 
cipitated by the addition of more CaCl 2 solution. 

234 






6. Dissolve the precipitate in 10.0 ml. of 0.5 N HC1. 

7. The optical density of the solution Is read at wavelengths of 380, 405 and 430 
mu,; v 0.5 N HC1 is used as the blank. 

Uroporphyrin Calculations : 

The uroporphyrin calculations follow the same pattern as those for the copropor- 
phyria Thus, the following steps are used in calculating the total micrograms of 
Uroporphyrin and the total microgram per cent. 

,„ 2D 405 ' < D 380 >* D430> n 

(1) _ = D corr 

(2) — £°£E x ml. acid solution = total micrograms uroporphyrin per ml. of 

065 original sample 

Since only 1 ml. of the original sample was used: 

(3) Total micrograms uroporphyrins x 100 = microgram per cent uropor- 

phyrins 

The total urine volume, total micrograms uroporphyrin and the total microgram 
per cent uroporphyrin should be reported. 

Interpretation : 

The normal amounts of coproporphyrin and uroporphyrin excreted dally Is small, 
50-300 micrograms and 0.5 micrograms respectively. However, in certain disease 
conditions abnormal amounts of these porphyrins appear. 

Coproporphyrin I and HI occur normally in human urine in minute and approximate- 
ly equal amounts. But, in a variety of diseases the excretion of these porphyrins is 
greatly increased. Also, the ratio of the I to the ni isomer may vary greatly, depend- 
ing upon the disease condition. For example , in poliomyelitis and lead poisoning 
coproporphyrin III Is excreted while fax infectious hepatitis coproporphyrin I predom- 
inates. 

The excretion of uroporphyrins Is characteristic of the metabolic disease of por- 
phyria. The photosensitive or congenital type is characterized by the excretion of 
uroporphyrin I while acute porphyria Is characterized by the excretion of uroporphy- 
rin m. 

Exact and reliable data relating porphyrin concentration to specific diseases con- 
clusively is not available. However, the abnormality of the porphyrin concentration, 
when coupled with other symptoms will help complete the diagnostic picture. 



735 



PROTKIN - (Turbiellmclrlc) 
Spinal Fluid and Urine 

References : 

1. Looney, J.M. and Walsh, A. I.: J. Biol. Chcm. 127 , 117 (1939). 

2. Henry, R.J. , Sobcl, C, and Scaglove, M.: Proc. Soc. Exp. Biol. Med. 
92, 748 (1956). 

Principle : 

Protein In spinal fluid or urine Is precipitated by trichloroacetic or sulfoaallcyllc 
acid as a-.fine uniform suspension. The turbidity thus produced Is measured photo- 
metrically. 

Reagents : 

Sulfoaallcyllc acid, 3 per cent solution or 
Trichloroacetic acid, 3 per cent solution 
(Keep refrigerated) 

Procedure : 

1. Into a test tube place 1. Ou ml. of cerebrospinal fluid. Add 4. ml. of 3% 
sulfosalicylic acid and immediately mix by inverting several times. 

2. Allow to stand at room temperature for five minutes and read before 10 min- 
utes against a blank of distilled water using light of about 660 mu (red). 

Calculation: 

Interpolate on a calibration curve or chart. 

Standardization: 

Prepare dilutions of a previously standardized serum containing from 0. 1 to 3 mg. 
of protein per ml. of solution. Carry out the procedure above and plot the results. 

Notes : 

1. Since the degree of reproducibility depends upon many physical factors such as 
temperature, speed of mixing, etc. , the details of the procedure should be standard- 
ized as much as possible. 

2. Trichloroacetic acid produces more turbidity from globulins than from albumin 
(1. 2 to }) while sulfosalicylic acid produces more turbidity from albumin (2 to 1) 
(Ref. 2), For this reason a mixture of the two acids, 2 of sulfosalicylic to 4 of tri- 
chloroacetic has been recommended. 

3. The wavelength of 660 mu is advocated because any yellow color of spinal fluid 
or urine will not affect the optical density at this wavelength. 

4. At best, the degree of reproducibility is no better than £ 10% and at times as 
high as i 20%. 

Interpretation ! 

See p. 239 (spinal fluid) 

236 






PROTEIN - TOTAL AND ALBUMIN/GLOBULIN BATIO 

Reference : 

Gornall, Allen G. , Bardawell, C.J. , and David, M.M.; J. Biol. Chem. 177 . 
751 (1949). 

P rinciple : 

Serum proteins (total) and the albumin fraction (after globulin precipitation) are 
reacted with alkaline copper reagent (biuret reagent) to give a bluish-purple color which 
Is measured photometrically in the presence of the blue color of the excess copper. 

Apparatus : 

Matched test tube cuvets 19 x 150 mm. or 19 x 105 mm. 

Reagents : 

1. Biuret reagent: Into a 1000 ml. volumetric flask place: 

CuS0 4 -5H 2 1.5 g. 

NaKC 4 H 4 6 -4H 2 (Rochelle Salt) 6.0 g. 
1.0 g. KI (optional) 
Dissolve in approximately 500 ml. Add and mix rapidly 300 ml. 2.5 N NaOH 
prepa -ed frcm concentrated NaOH; see stock reagents). 
Add water and mix to make 1000 ml. 

2. Sodium sulfate 22. 6%. Dissolve 226 g. of the best quality anhydrous Na SO. 
In water to make 1000 ml. of solution. 



3. Ethyl ether 

4. Sodium Chloride 1% 






Procedure : 

Total Protein : 

Into a dry test tube which can be centrifuged, measure 0.50 ml. of serum, and 
add 10.0 ml. of 22.6% Na„S0 4 solution. Stopper the tube, and mix thoroughly by inver- 
sion (not shaking). At once , transfer 2.0 ml; to photometer tube T for total protein. 
Albumin (/ * l globulin) 

To the remaining serum -sodium sulfate mixture, add 3 ml. of ether, shake 
vigorously for 30 seconds, and centrifuge while still stoppered; slant the tube, and 
transfer 2.0 ml. of the clear, subnatant aqueous phase to a dry photometer tube A for 
the albumin determination. 
Blank : 

Into a dry photometer tube B, place 2.0 ml. of 22.6% Na 2 S0 4 solution. 

To each of these three photometer tubes add 4.0 ml. of biuret reagent, and 
mix well. Allow the tubes to stand for 30 minutes at room temperature (20-25° C). 

Read in the photometer against the blank set at 100% transmission using wave- 
length 550 mu. From the transmission in % determine the density (transmission vs. 
optical density chart) and refer this value to a calibration curve prepared as described 
below or multiply the density by a factor. 

237 



Procedure for total protein without albumin determination: 

1. Plank : Into photometer tube B measure 2.0 ml, 1% NaCl solution. 

2. Total protein: Into photometer tube T measure 2,0 ml. of 1% NaCl solution. 
Pipet into this 0. 100 ml. of serum with a "to contain" micropipet. 

3. To each of the photometer tubes add 4.0 ml. biuret reagent and mix well. 
Let stand 30 minutes at 20-25 C. and read in photometer as above.. Jtefer to 

the same calibration curve; since the volume in this case is 6. 1 ml. lastead of 6. 
ml. the final results for the simplified total protein should be multiplied by 81/60. 

Calibration : 

Pipet 5. 00 ml. of clear normal (preferably pooled) serum Into a 60 ml. volumetric 
flask, dilute to mark with 0.9% NaCl, and mix. Prepare In duplicate a series of nine 
photometer tubes as follows: 

0.9% (a) (b) (c) 

Tube » ml. NaCl ml. dil. serum fV) (V x P) 

0.00 0.00 0.00 

0. 16 0. 1576 

0.30 0.3150 

0.45 0.4725 

0.60 0.630 

0.80 0.840 

1.00 1.060 

1.20 1.260 — *~ 

1.40 1.475 

Develop color in each tube by adding 4.0 ml. biuret reagent as above. 

(a) Serum diluted 1 volume up to 10 volumes with 0. 85% NaCl 

(b) Column (a) corrected for 1:21 Instead of a 1:20 dilution of unknowns 

(c) Values to be plotted vs. optical density, "nominal" protein concentration. 
P r protein concentration of the original serum as determined by 
Kjeldahl procedure 

Determine total nitrogen and non-protein nitrogen and multiply protein nitrogen by 
6. 25 to obtain protein in g. %. 

The resulting graph gives a practical straight line and therefore a Beers Law factor 
may be used with a maximum error of 0. 1-0. 2 g. % protein. 

Notes : 

1. For room temperatures above 25°C. - recalibrate. 

■ 

Interpretation : 

1. A variety of different precipitating salts have been used. With this present 
method albumin Is usually 4. 5 g. %; globulin 2-2. 6g%; total protein 6-7. 5 g. %. A 
total salt concentration higher than this gives lower "albumin" and higher globulin. 



236 



( 



1 


2.00 


2 


1.85 


S 


1.70 


4 


1.55 


6 


1.40 


8 


1.20 


7 


1.00 


8 


0.80 


9 


0.80 






PROTEIN 
Spinal Fluid - Biuret Mtlioil 

Reference 

1. Rosenthal, H.L. and Cundiff , H.T.*. Clin. Chcm. 2, 394 (1956) 

Principle: 

Ethyiencdiamine tetraacetate la added to the biuret reagent (in place of tartrate) 
to avoid turbidities due to calcium when the necessarily larger volumes of spinal fluid 
are used. 

Reagents : 

1. Stock protein standard 

A standardized serum (or serum albumin) is accurately diluted with 0. 85% NaCl 
to about 50 mg. protein per milliliter. This may be stored about 30 days at 4°C. or 
indefinitely at -20°C. 

2. Protein working standard 

Dilute 5.00 ml. protein stock solution to 100 ml. with 0.85% NaCl. Prepare on 
day of use . 

3. EDT A- biuret reagent 

Dissolve 1.50 g. CuS0 4 - 5H 2 in about 500 ml. distilled water. Add 6.0 g. 
disodium ethylene diamine tetraacetate (NagEDTAJand 1. g. KI and dissolve. Add 
300 ml. 2.5N NaOH, mix well and dilute to one liter. Store at room temperature in 
polyethylene containers. 

Procedure : 

Blank - 2. 00 ml. 0. 85% saline 

Standard - 2.00 ml. protein working standard 

Unknown - 2.00 ml. cerebrospinal fluid 

Add 4.00 ml. of EDTA-biuret reagent and mix well. 

Allow to stand 20-30 minutes at room temperature, and then compare standard 
and unknown against the blank in a photometer set at 550 mu. 

Calculation : 

(Du/ D ) C x J5* - mg. protein/100 ml. spinal fluid 
8 2 

„ . C a >■ mg. protein present hi 2.00 ml. of protein working standard. 

1. A series of standard protein solutions may be used to prepare a standard curve 
instead of using daily standards. 

2. If the protein value exceeds 300 mg. /100 ml. the sample should be diluted and 
the analysis repeated. 

3. H desired, the same reagent may be used for serum protein analysis; however, 
the sensitivity of the reagent Is somewhat less. 

Interpretation: 

Normal - lumbar spinal fluid - 15-40 mg.% total protein - almost entirely albumin 
Globulins may be detected by the Pandy test, 

239 



PROTHROMBIN TIME 
(One -stage Quick Method) 



Reference: 



Quick, A. J., Am. J. Clin, Path. 10, 222 (1940) 






Principle : 

Oxalated plasma Is treated with an excess of thromboplastin and an optimum con- 
centration of Ca^. The clotting time Is determined and related to the pro-thrombln 
content of the plasma, as shown In the accompanying representative graph (A) of clot- 
ting times vs. plasma concentration (in 0. $5% saline). 









70 
60 
50 
40 

30 

20 

10 























-T Xv 


















» 1 




A 














- B 
















v 
















' 






. , i 


_j . 


i - 


■ 


i 


i 


i 


l 





10 20 30 40 50 60 70 80 90 100 
% plasma 
Obviously dilution of plasma in saline (curve A) causes a dilution of not only pro* 
thrombin but also of all other clotting factors, except thromboplastin and Ca^r which 
are kept constant. If normal plasma is diluted in plasma previously treated by BaSO^ 
(which removed pro-convertin and prothrombin) and clotting times again determined 
curve B is obtained. The difference between the two curves represents the combined 
effect of factors other than prothrombin and proconvertin. Curve B Is the combined 
dilution curve of prothrombin and proconvertin. 

Since in dlcomnarol and hedulln therapy for thrombosis both prothrombin and procon- 
vertin are decreased, curve B represents the usual therapeutic situation. 

The only other factor likely to affect the results is pro-acceleria and since this Is labile, 
collection problems and delay in analysis may affect this markedly. This factor can be 
added in excess by the use of BaSO^ plasma. 

In this laboratory we run both the regular Quick one -stage procedure and the regular 
procedure modified by the addition of BaSO, plasma. We call these tests "uncorrected" 
and "corrected". The "corrected" test Is run only when pro-accelerln deficiency or lack 
is indicated. 
Reagents : 

1. Plasma. By careful venipuncture collect exactly 4.5 ml. of whole blood. 
Mix in centrifuge tube with exactly 0. 5 ml. of 0. 1 molar sodium oxalate 
(1.34 g. of anhydrous sodium oxalate in 100 ml.). Centrifuge at once for 



( 



240 



4 minutes at 2,000 r.p.m. Plpct off the supernatant plasma, avoiding any re- 
mixing of red blood cells. Use within a short time. If there is to be a delay 
before determining prothrombin activity, keep plasma chilled until it is to be 
used. Warm chilled plasma in water bath for 5 minutes before use, or 15 min- 
utes in warm room, 

2. Thromboplastin. 

3. 0.0125 MCaCl 2 (calcium chloride). 

4. Dilute pro-accelertn plasma (see Preparation of Reagents below). 

Procedure : 

1. Quick procedure ("Uncorrected") - Warm all reagents to 37° C. before use. 
Conduct tests at 37° C. 

a. Using 0. 1 micro-pipets, mix 0. 1 ml. of thromboplasin and 0. 1 ml. of 
calcium chloride solution In a small test tube, approximately 10 x76 mm. 
Keep in water bath or air bath, at 37° C. 

b. Using 0. 1 ml. mlcro-pipet, blow 0. 1 ml. of warmed plasma Into the 0.2 ml. 
of thromboplastin-calcium mixture. Mixing of reagents should be rapid and 
complete. 

c. Start timer simultaneously with addition of plasma. 

d. Stop timer when end-point is reached. The end-point Is the first appearance 
of a clot as determined by visual observation while (1) tapping the tube with 
a finger, (2) tilting the tube, or (3) drawing a loop of #20 nichrome wire 
through the coagulation mixture or (4) by a photoelectric recorder, 

e. The prothrombin time thus obtained has no absolute meaning but must be 
converted into terms of prothrombin activity by reference to a previously 
established normal plasma dilution curve , or a table prepared from it (see 
below). 

2. Quick procedure ("Corrected^ - Using 0. 1 ml. and 0. 2 ml. pipets mix 0.2 ml. 
thromboplastin-calcium and 0. 1 ml. of dilute pro-accelerin plasma, Incubate at 
37° C. for 5 minutes. Then add 0. 1 ml, of the test plasmaand start timing by 
one of the methods listed above. 

Notes and Interpretation: 

The determination of the blood prothrombin level is of great importance in the diag- 
nosis of hemorrhagic states due to Vitamin K deficiency, biliary obstruction and liver 
damage. The hypoprothrombinemia produced by dicoumarol In the prophylaxis and 
therapy of Intravascular thrombosis requires frequent prothrombin determinations in 
order to guard against spontaneous hemorrhage. 

The optimal concentration of calcium required for prothrombin determination is influ- 
enced by the amount of oxalate used In collecting the blood sample. This is especially 
true for the one-stage method. Quick's directions foroxalating 4.5 ml. of whole blood 
with 0.5 ml. of 0. 1 molar sodium oxalate must be adhered to rigidly. Some laborator- 
ies use citrate or EDTA. When changes are made, the optimal Carr concentration 
must be redetermined. The fibrinogen level of plasma Is usually of little consequence 



241 



PHOTHKOMDIN TABLE 

Values in the body of the table represent the ratio between 

the clotting tlmefl of the unknown to the control plasma Usee. 






















C 


Bee. 




% 

Prothromblr. 


l ° 


1 


2 


3 


4 


5 


6 


7 


6 


9 





_ 


„ 


M 


*. 


_ 


wm 


5.600 


4.96 


4.320 


3.660 


10 


3.040 


2.94 


2.84 


2.74 


2.65 


2.55 


2.46 


2.36 


2.27 


2.17 


20 
30 


2.080 
1.680 


2.04 
1.66 


2.00 
1.64 


1.96 
1.61 


1.92 
1.69 


1.88 
1.56 


1.84 
1.54 


1.80 
1.51 


1.76 
1.49 


1.72 
1.46 


40 


1.440 


1.410 


1.390 


1.380 


1.360 


1.350 


1.330 


1.320 


1.310 


1.290 


50 


1.280 


1.270 


1.260 


1.251 


1.241 


1.231 


1.222 


1.212 


1.203 


1.193 


60 


1.184 


1.177 


1.171 


1.165 


1.158 


1.152 


1.146 


1.139 


1.133 


1.126 


70 


1.120 


1.115 


1.110 


1.106 


1.102 


1.098 


1.094 


1.090 


1.086 


1.081 


80 


1.077 


1.073 


1.069 


1.064 


1.060 


1.056 


1.052 


1.048 


1.044 


1.039 


90 


1.036 


1.032 


1.029 


1.025 


1.022 


1.018 


1.014 


1.011 


1.008 


1.004 


100 


1.000 




















Calculation: 


(UBlUg 


ratio method) 

















1. Calculate the ratio Unknown time (sec.) /Control time (sec.) 

2. Find value closest to this ratio in. the table. 

3. Head prothrombin % directly. 

Example: Control time = 16.3 sec. Uncorrected 

a 15.2 sec. Corrected 

Unknown time = 17.2 sec. Uncorrected 

= 15. 8 sec. Corrected 

Uncorrected Corrected 

17.2 .. 1.056 =- 85% IS- 8 - 1.040 - - S9% 

16.3 15.2 

It is realized that these "percentages" of normals do not actually represent the 
patient's prothrombin concentration. However, clinicians are accustomed to treating 
patients on the basis of this dilution curve of Dr. Quick and we continue to use It as 
a matter of convenience. The more accurate procedures are also somewhat more 
time consuming. 



242 



TABLE FOR DETERMINING PROTHROMBIN PERCENTAGES 
(Maura 1 of Clinical Biochemistry - W. G. Karr, 
J. G. Reinhold, and F. W. Chornock - p. 49) 



Control 






Prothrombin, 


Per cent of Control 








100 


85 


70 


60 


50 


40 


30 


20 


10 


6 










Seconds 










12.5 


13.2 


14 


14.8 


16 


18 


21 


26 


38 


70 


13.0 


13.7 


14.5 


15.4 


1G.G 


18.7 


21.8 


27 


40 


73 


13.5 


14.3 


15.1 


16.0 


17.3 


19.4 


22.7 


28 


41 


76 


14 


14.8 


15.7 


16.6 


18 


20.2 


23.5 


29 


43 


78 


14.5 


15.3 


16.3 


17.2 


18.6 


20.9 


24.4 


30 


44 


81 


15 


15.8 


16.8 


17.8 


19.2 


21.6 


25 


31 


46 


84 


15.5 


16.4 


17.4 


18.4 


19.8 


22.3 


26 


32 


47 


87 


16 


16.0 


17.9 


19 


20.5 


23 


27 


33 


49 


90 


16.5 


17.4 


18.5 


19.5 


21.1 


23.8 


27.7 


34 


50 


92 


17 


18 


19 


20.1 


21.8 


24.5 


28.6 


35 


52 


95 


17.5 


18.5 


19.6 


20.7 


22.4 


Aii>. 2 


29.4 


36 


53 


93 


18 


19 


20.2 


21.3 


23 


26 


30.2 


37.5 


55 


101 


19 


20 


21.3 


22.5 


24.3 


27.5 


31.9 


39.8 


58 


106 


20 


21 


22.4 


23.7 


25.6 


29 


33.6 


42 


61 


112 


21 


22.2 


23.5 


24.9 


26.9 


30.2 


35.3 


44 


64 


118 


22 


23.2 


24.6 


26 


28 


31.7 


37 


46 


67 


123 


23 


24.3 


25.8 


27.2 


29.4 


33.1 


39 


48 


70 


129 


24 


25.4 


26.9 


28.4 


31 


34.6 


40 


50 


73 


135 


Calculation: 



















Example: Normal Control time Is 14.5 seconds. Patients time Is 18.0 seconds. 
Then prothrombin % is between 60 and 50%. By Interpolation: 

IB. 6-18. o JK_6 m o.428; (0.428 x 10) / 50 = 54.3% 
18.6-17.2 1.4 












243 



In the one-stage method, since considerable range of fibrinogen content appears to 
have little effect on the prothrombin time. 

The lability of accelerator-globulin (labile factor) (accelerin or pro-acceierin) Is fre- 
quently a source of error in the one -stage method. The handling of the plasma in this 
method has a distinct effect on the adequacy of Its accelerator-globulin content. For 
this reason, the blood sample should not be exposed to extremes of centrlf ligation, 
either in intensity or duration. A short, light centrifugation is essential for the preser- 
vation of accelerator-globulin activity. Since accelerator -globulin is heat-labile, the 
plasma must not be incubated for long periods of time. Plasma should be tested for 
prothrombin activity within a short time after its collection or else should be chilled 
until needed for use. 

The final factor required for prothrombin activity determination 1b thromboplastin. This 
factor must be present in excess. To a large extent the validity of the measurement of 
prothrombin activity is dependent upon the thromboplastin. The principal requirement 
for a satisfactory thromboplas tic material is that it yield accurate and consistent pro- 
thrombin times. When all other factors have been controlled by careful, uniform tech- 
nique, the prothrombin times obtained measure prothrombin activity only when the 
thromboplastin used gives reliably reproducible values for given prothrombin concen- 
trations. Other requirements of a good thromboplastin are stability and convenience. 

Proper handling of thromboplastin requires only a few precautions. Although thrombo- 
plastin is stable at room temperature for at least two weeks, it Is advisable to store 
the material In the refrigerator at about 4° C. when not in use. The usual routine Is 
to withdraw from the stock bottle a volume of thromboplastin sufficient for immediate 
requirements and to replace the stock bottle in the refrigerator. Care must be taken, 
however, to avoid freezing the solution. 

If several prothrombin determinations are to be made, It is more convenient to mix 
equal volumes of thromboplastin and calcium chloride solution. 0. "2 ml. portions of this 
thromboplastln-calcium mixture are pipetted into the required number of tubes and 
kept in the water bath ready for use. The test is then continued as described below. 
Discard unused mixture of thromboplastin-calcium at end of the working day. The 
order of addition of reagents as given in the directions must be followed. Changing 
the order by mixing plasma and thromboplastin and then adding calcium chloride solu- 
tion sometimes gives erratic results, particularly If the mixture Is incubated for any 
length of time. Reproducibility of accurate prothrombin times depends on uniformity 
of a technique. Only by careful observance of uniform procedure in the performance of 
the test can maximum accuracy be obtained. 

Prothrombin times are used clinically primarily for control of anti-coagulant therapy 
in patients who have (a) phlebitis (b) coronary occlusion (c) any other disease which 
tends to produce intra -v as cular clotting. The prothrombin level should be kept be- 
tween 15% and 20% of normal although different clinicians and hospitals vary. Bleeding 
tendencies begin to show up at 10% or below in many patients. 

244 



PREPARATION OF PLASMA PROTHROMBIN REAGENTS 



Preparation of pronccelertn plasm a: 

1. Collection of plasma: Collect by the use of vaculamer tubes by heart puncture 
more than 100 ml. of oxalaled plasma with the oxalate concentration recom- 
mended by Quick (ref. clt.). 

2. Teat lor the presence of prothrombin and proconvertin by the following tests, 
which also should be run after each adsorption (see (3).) 

Tests for presence of prothrombin and/or proconvertin* 

Test System A: 0.2 ml. thromboplastln-calcium 
0. 1 ml. rabbit plasma 
Measure clotting time 
Results: In the original rabbit plasma the time may be as short as 

6 seconds. When treated with BaS0 4 the time will be pro- 
longed and should be over 150 seconds. However, this may 
not represent a complete removal of prothrombin but mere- 
ly a removal of proconvertin since'the latter Is preferen- 
tially removed before prothrombin. Therefore Test Sys- 
tem B must be used. 

Test System B: 0.1 ml. aged human serum 

0.2 ml. thromboplastln-calcium 
Incubate 5 minutes 
0. 1 ml. rabbit plasma 
Results: The aged human serum contains no prothrombin (all has 

been converted to thrombin and adsorbed and neutralized) 
but does contain pro-convertin; now if the rabbit plasma 
Is free of pro-convertin but still contains some pro-throm- 
bln clotting will occur. 

3. Add to 0. 1 g. wet BaSO. (washed twice with distilled water by suspension and 
centrif ligation) for each ml. of plasma, the volume of plasma obtained above. (1). 
Allow to stand at room temperature for 15 minutes resuspendlng the BaSO a at 
intervals. Centrifuge 10 minutes at 3000 r. p. m. (preferably in a refrigerated 
centrifuge) and carefully remove the supernatant plasma. 



Treatment with washed BaSO. should be continued until the time of clotting in 
test system A and B are prolonged to more than 150 seconds. 
Some samples of BaSO. are not very active in adsorption, and their activity may 
change with time. In this case, a new batch may be used or Quick's washed 
Ca„ (P0 4 ) 2 suspension may be used, or freshly prepared BaSO^ may be used. 
A. BaS0 4 To 8.5 ml. 1 M BaCl 2 add 8. 5 ml. 1 M Na 2 S0 4 (2 gm. BaS0 4 ). 

Mix, centrifuge, and resuspend in water to wash, recentrifuge . Repeat. 

Use the 2 gm. for 25 ml. of plasma. Allow to adsorb for 30 min. at room 

temperature. 

245 



B. Cajj (TC\)- 

Sol. A. 158 p. NaLgPC^- 123^0 to one liter HgO 
B. C6. 6 g./ CaClg snhyd. In one liter HgO 
Add SoL A to Sid. B slowly sod with Timorous stirring. AdjnstpH to 
7.0 by adding; BC1 or JfeOH. Wash by decaxdtatum nnta free of Cl~. 
(A£H0 3 / HHO^. Bring ▼plume to 1 Hter. This is a 0.02 M stock 
suspension. For dilate snsngBBton: Snake weB. Dilate 4 ml. / 9S ml. Had. 
For each mL of plasma, centrifuge 1 ml. of dilate sasnesgion, and discard 
the sopsrssle. Use as above. 

3. The aged hnraan serum le prepared as follows: Ten ml. of blood are Am Into 
aVacntainer, widen is then immedlEteJy opened. 0.2 ml, of thromboplastin 
(Solupte-stin) is added nnd the blood mixed well. Clotting; occurs rapidly and me 
tabs Is fbzn allowed to eft ct 37° for 30-40 minutes. Tbs sersm Is Gen re- 
mored end Is ready lor the test. Usually a dilution of 1:10 or 1:5 with water 
ess be made stace me serum contains large amounts of coxfcrtm. 

Test for Fro-ecceleria Actj vtly: 

When lbs pxev&cs tests A and B have Indicated tbe virtual absence of proc on ver ti n, 
and proiirorabfa from the rabbit platans. Its proaccelerm activity mast "be tested. For 
tins we need a p!2sia& free of or low ia pro-acceleria. This is o bta ined bj dr aw tag *■ 
sainpfe of blood Into a prothrombin Vacctalaer lobe and mixing. Oesfxi&xje and remove 
fheplassa. Allow to remain at 37° until tbe "Oacorrected"Qaick prothrombin time la 
prolocged to at least 30 seconds. 

( 
Then, using Test System C. determine bow far the rabbit plasma (Free of pro c onv e rt in 



and prothrombin) can be (filmed and stOl retain Its uminmra activUj. 

Test System C. 

0.2 mL thromboplastln-calctnm 
0.1ml rabbit plasma 
Incubate 5 min. 
0.1 mL aged human plasma 

Usually dog plasma can be diluted at least 1:4 and r*bhit plasma 1:8 before a noticeable 
decrease In activity is seen. 



When tbe greatest dilution possible has been eBtahllsnea, dilute the bulk of the rabbit 
plasma in water, mixweB, adjust the pH to T.4, and freexe to 2 ml. portions at 
-20°C. Use ove or two portions daUy. Doi 









246 






Preparation for coagulation tubes : 

After use, soak in a 2% solution of Na^COn 5 houra or overnight. Rinse well with 
tap water and then with distilled water and bake at more than 110° C. overnight. If 
the tubes arc siliconized, removal of the clots is somewhat simplified. 



Directions for Siliconizing: 

Silicone - One part Dri-Fllm (General Electric) #9987 is added to four parts 
Petroleum Ether. 

Fill one tube with this mixture and pour Into another rotating the first tube so that 
the entire inside area is covered. Avoid siliconizing the outside since the tubes will 
be too slippery to handle easily. Rinse the tubes (in a rack for convenience) with hot 
tap water, allowing the water to remain for about 10 minutes. This step removes any 
silicone flakes and speeds the removal of the HC1 base of the drl-film. Rinse twice 
with distilled water and dry at 120° C, for 60-90 minutes. 

To remove silicone - Put the tubes In a mixture of 2 1. distilled H„o(ln which 150 
gms. NaOH is dissolvedjand 2 1. of technical acetone. A How to stand for at least 8 
hours and rinse well after removing from the solution, dry at 120° C. Repeated re- 
moval of silicone by this solution will result In etched tubes that will not look clean, 
however, upon being resillconized, the tubes will be entirely clear and the clots 
easily read. We suggest that you keep these tubes separate from the others. 






247 



PROTHROMBIN 
{Prothrombin Consumption Test) 
Scrum 
Reference : 

Leon N. Sussman, M.D. , Ira B. Cohen, M.D. , and Robert Glttler. M.D. 
J. A.M. A. 15G, 673-752(1954). 

Technique : 

Careful attention must be paid to details In technique, as minor variations pro- 
duce very marked differences in the results obtained. 

Preparation of Serum : 

Blood is drawn cleanly and allowed to clot at room temperature, The blood Is 
placed in a water bath at 37° C. for one hour after clotting. The blood Is centrl- 
fuged for three minutes, and the serum is separated. Serum maybe stored at 4° C. 
for a maximum of 60 minutes before the test. 

Preparation of Reagents: 

Thromboplastin solution Is prepared as for the plasma prothrombin test. Fibri- 
nogen solution is prepared as directed by the manufacturer (to contain 300 mg. per 
100 cc. of fibrinogen and 85% of sodium chloride solution). Two cubic centimeters 
of thromboplastin solution and 1 cc. of fibrinogen solution are mixed Just before the 
test. 

Performance of Test: 

The mixture of thromboplastin and fibrinogen, in the amount of 0. 2 cc. is placed 
in a test tube in a water bath at 37° C. for five minutes. The serum to Detested is 
warmed in the water bath at 37° C. for five minutes. One-tenth cubic centimeter of 
the serum Is blown strongly into the thromboplastin-fibrinogen mixture ( the timer 
should be started simultaneously) , and the time for the clot to form Is determined 
as in the plasma prothrombin test. 

The clot is formed as a fine web, and the person performing the test should watch 
carefully for It. There are other precautions to be observed. The serum must be 
refrigerated at 4° C. for a maximum of 60 minutes if It is not used immediately. 
The thromboplastin and fibrinogen must be mixed Just before use. All reagents must 
be warmed at 37° C. for five minutes before use. The test is interpreted as follows: 
time longer than 30 seconds. . .normal range; time shorter than 20 seconds. . .abnor- 
mal range; and time between 20 and 30 seconds. . . doubtful. 

Note: 



( 



BaSO . plasma entirely devoid of prothrombin and pro-convertin may be used 
instead of fibrinogen. 



( 



248 



SALICYLIC ACID 
Scrum or Plasma 

Reference : 

Bernard B, Brodle, Sidney Udenfriend, and Alvin F. Coburn: J. Pharmacol. 
Exp. Therapeutics 80, 114-117 (1044) (Modified). 

Reagents : 

Salicylic acid solution, stock standard, 100 mg. per 100 ml. Dissolve 116 mg, of 
sodium salicylate (U.S. P. cryst. Merck) In water and dilute to 100 ml. Mix. Stable 
when stored in refrigerator. 

6 N HC1, made by diluting concentrated HC1 with an equal volume of water. 

Ethylene dichloride (Ethylene Chloride, Eastman #132). 

1% Fe(N0 3 ) ? - BH 2 in 0.07 N UNO,. Dissolve 1 g. of ferric nitrate, A.C.S. ill 
water. Add 0. 44 ml. of concentrated nitric acid and dilute to 100 ml. Mix. This Is 
a stock solution. 

Dilute ferric nitrate solution. Add 3 ml. of above stock solution to 100 ml, of. 
distilled water. Mix. Keeps a few days. 

Sodium sulfate , reagent anhydrous. 

Procedure : 

Into a clean dry glass stoppered 60 ml. bottle place 1 ml. of serum or plasma. 
Add 0. 2 ml. of 6 N HCl and 15 ml. ethylene dichloride. Shake 5 minutes, preferably 
by machine. Add approximately 1 g. anhydrous sodium sulfate. Shake 1 minute. Pour 
the organic layer into a tube, stopper with a cork, and centrifuge until clear. 

Remove 10 ml. to another clean, dry bottle. Add 15 ml. of dilute ferric nitrate 
solution. Shake 5 minutes. Transfer 10 ml. of the aqueous layer to a cuvet. Read In 
a photometer (515 mu) using distilled water for a blank. 

Calculation : 

Construct a standard curve by analyzing 1 ml. quantities of aqueous salicylate solu- 
tion of varying concentrations, by the above procedure. 

Interpretation : 

Satisfactory plasma levels for treatment of rheumatic fever are 36 to 40 mg.% of 
salicylic acid. See Alvin E. Coburn: Bull. Johns Hopkins Hosp. 73, 435-464 (1943). 
ft is important to use the chemical analysis to control therapy, since the therapeutic 
dose is very close to the toxic level. 

Note : (1) Save the ethylene dichloride solutions for recovery by distillation. (2) By this 
modification the absolute extraction of salicylic acid is only 95.8% complete but since 
standards and unknowns are treated in an identical manner, 100% recoveries are obtain- 
ed. 



249 



falODIUM AND POTASSIUM 
St* rum, U rine and TisEue 

References : 

1. Barnes, R. B., Richardson, D. Berry, J. W, , Hood, .R. L. : Ind. Eng. Chem. 
Anal. Ed., 17, 605 (1945). 

2. Berry, J. W. , Chappell, D. G. . and Barnes, ii. B. , ibid., Jjg, 19 (1946) 

Principles of Flame Photometry: 

In flame photometry, an aqueous sclution Is atomized under reproducible condi- 
tions by some sort of spraying apparatus. The vapor U conducted to the air intake of a 
gas burner and ignited. At the relatively low temperature of a gas burner, most ele- 
ments are not energized to emission teriperature and only sodium, potassium (and to 
a lesser degree calcium and magnesium) emit light. 

The Intensity of the light emitted is measured by a photocell with an appropriate 
filter combination (a prism or grating s >ectrophotometer may also be used to isolate 
the light wavelengths of interest). By measuring the Intensity of light given by solu- 
tions containing known concentrations, r curve can be obtained which .relates the In- 
strument reading to concentration. Thlt is known as the "Dire ct" method. 

Many factors influence the Intensity "of light emitted and n easurec. using a solution 
of constant composition. Some of these ire listed. 

1. Gas flow rate 

2. Air flow rate 

3. Temperature of flame 

4. Rate of atomization 

5. Efficiency of atomization 

6. Presence of other ions (anions and cations) 

7. Efficiency of filters, or of the pi Ism or grating usedtt isolate wavelengths of 
light. 

8. Stability of light measurement ssstem (photocells) amplifier 4 .*. and galvan- 
meter 

9. Purity of air supply (if burner supply is atmospheric). 
Some of these factors can be controlled, some are inter-related, others may vary 

erratically or are uncontrollable. The "Internal Standard" was developed to minimize 
the effect of variation In these factors. 

In the "Internal Standard" procedure, a known concentration of a salt containing an 
element with an emission line different from the element being determined, is added to 
the standard and to the unknown series of solutions. Lithium salts are commonly used. 
In this method the lithium emission Is affected to the same extent as that of the element 
being determined by most of the foreign influences listed above. Instead of measuring 
direct Intensities, the ratio of the Intensities of light produced by (e.g.) sodium to 
lithium Is measured, and the ratio Is not affected by the variable factors, 1-6. 



250 









Reagent h ; 

Stock lithium solution 2 N 
L1CI 8-1.80 g. /liter 
or LINO 137. 00 g. /liter 

or L1 2 C0 3 ( / HN0 3 to dissolve) 73.89 g. /liter 

Dilute LI solution: 20 ml. of stock diluted to 1 liter with distilled water 
Stock mixed sodium and potassium, standard 
KC1 (dry) 0.4475^. 



1. 



NaCl (dry) 9. 352G g.) 



Diluted to 1 liter with dlBtilled water 



Tl; 3 makes a solution 160 meq, Na and 6 meq. K per liter 
4. Dilute mixed standard - see under Procedure 

Procedure : (using the Baird flame photometer) 

1. Preparation of serum : 

Collection should be made in plain tubes, avoiding rough handling and hemolysis 
which will release potassium into the Eerum. Merely allowing serum to stand over 
cells for prolonged periods will cause high serum K values. 

2. Lighting and adjusting burner : 

a. Turn on air at stopcock, 

b. Turn air regulator control clockwise until 5 lba./sq. Inch Is registered on 



the dial. 



c. Turn on gas and light burner at top with lighted match. 

d. Increase air pressure to 10 lbs./sq. inch. 

e. Allow apparatus to warm up 15-20 minutes before calibration. 



Sodium Standardization 

1. Preliminary adjustment 

a. Rotate the filter wheel to the Na position. The filter wheel must CLiCK 
into position. 

b. Set the SENSITIVITY knob to the indicated mark ^bout 10% of full rotation), 

c. Set the BALANCE control at 700. 

d. Set the selector SWITCH at DIRECT. 

e. Using the galvanometer knob, adjust the galvanometer to read zero at the 
center of the top scale, thus: 

ill! 




2. Standardization 



a. Set the selector SWITCH to EXTERNAL STANDARD 

b. Pour dilute mixed standard into the funnel. 

c. Adjust the SENSITIVITY >to return the galvanometer to zero, 
point, DO NOT READJUST THE SENSITIVITY! 

d. After the funnel has run drv, set SWITCH to DIRECT. 



(After this 



251 



e. Using the galvanometer knob, adjust the galvanometer to read zero, 

f. Reset the SWITCH to INTERNAL STANDARD. Pour more dilute ml»»d 
standard into the funnel and adjust the BALANCE control to zero the ealvanometer. 

g. Repeat steps d, e, and f until It is not necessary to make any adjustment to 
zero the galvanometer when 

(1) the funnel is dry and the SWITCH at DIRECT 

(2) the funnel is full of dilute mixed standard and the SWITCH at INTERNAL 
STANDARD. 

Potassium Standardization : 

1. Preliminary adjustment 

a. Rotate the filter wheel to the K position. 

b. Set the BALANCE control at 250. 

c. Set the SENSITIVITY knob at the indicated mark (about 60% of lull rotation), 

d. Set the selector SWITCH at DIRECT. 

e. Using the galvanometer knob, adjust the galvanometer to read zero at the 
center of the scale. 

2. Standardization: 

Carry out steps a-g as .under sodium standardization. 

Procedure for 1.00 ml. serum: 

1. Prepare dilute mixed standard by rinsing out a glass -stoppered 100 ml. vol- 
umetric flask with dilute lithium solution at least three times 

2. Pipet 1. 00 ml. of stock mixed standard into the flask. 

3. Dilute to the mark with dilute lithium solution and mix well by repeated inver- 
sion with shaking. 

4. Prepare the serum dilution in a similar manner using 1.00 ml. serum. 
6. Compare the serum dilution with the dilute standard as outlined below. 

Procedure for . 200 ml. serum: 



1. Prepare dilute mixed standard by rinsing out a glass -stoppered 100 -ml. vol- 
umetric flask with dilute lithium solution at least 3 times. 

2. Fill to the mark with dilute lithium solution. - 

3. Add to thisflOO ml. of dilute lithium solution^ 1. 00 ml. of stock mixed standard. 
(This results in a 1:101 dilution of the standard.) 

4. Into a dry 50-ml. Erlenmeyer flask pipet 20.00 ml. of dilute lithium solution. 
With a "to contain" 0. 20 ml. blood pipet, add 0.200 mi. serum, rinsing back and forth 
at least eight times. Care should be taken to avoid touching the glassware with the 
fingers since perspiration Is very high in Na and K. This results in a 1:101 dilution 

of serum 

5. Compare the serum dilution with the standard as outlined below. 



252 



Comparison of Standard and Unknowns : 

1. Standardize the Instrument for sodium or for potassium as outlined above. 

2. Pour dilute mixed standard into the funnel and adjust the BALANCE control to 
zero the galvanometer. Record the dial reading R s . 

3. After the standard has passed through the aspirator, pour some of the serum 
dilution Into the funnel, and again adjust the BALANCE control to zero the galvanometer. 
Record the reading R u . 



Calculation: 



Example: 



<R U /R a > x C„ - C u 



Na Jt 

R 8 = 710 R B : 266 

R u = 606 R^ = 207 

The standard contained 160 meq Na and 6 meq K per liter. 
Na (606/710) x 160 = 137 meq Na/llter. 
K (207/256) x 6 = 4. 85 meq K/llter 

■ 

Interpretation : 

See under Electrolytes (Appendix p. 321) 

Note : Exact amounts of lithium to use and exact setting 6f the BALANCE control will 
vary with the sensitivity of the photocells. 



253 



SULFA DRUGS 
Blood, Urine and Spinal Fluid 



c 



Reference : 

Bratton. A.C. and Marshall. E.K.: J. Biol. Chem. 128, 537-550 (1939). 

Principle : 

The drug is dlazotlzed and, after excess nitrous aci d has been destroyed by 
sulfamic acid (HNO V HOS0 2 NH 2 . H 2 50 4 / HO / N 2 ), Is coupled with N-(l-naphthyl)- 
ethylenediamine dihydrochlorlde to form a colored product (an azo dye). 



Reagents: 

Saponin, 0.05%. 0.5 g. In 1000 ml. of water. 

Trichloroacetic acid (TCA), 15% In water and 3% In water. The U.S. P. grade Is 
satisfactory. 

Sodium nitrite, 0. 1% In water. Make fresh from 50% NaNO„ (refrlg.). 

Ammonium sulfamate (NH 4 OS0 2 NH„) , 0.5% In water. Make fresh. 

Coupling reagent. N-(l-naphthyl) -ethylenediamine dihydrochlorlde, 0.1% In 

. Fresh dally. 

Stock standard sulfanilamide. Weigh accurately 0. 1000 g. of sulfanilamide, 
transfer quantitatively to a one-liter volumetric flask, dissolve In hot water, cool to 
room temperature, dilute to volume, and mix. 1 mi, 3 0. 1 mg. This solution will 
keep several months In a refrigerator. 

Dilute standard. Into a 100 ml. volumetric flask containing 18 ml. of 15% TCA 
pipet 3 ml, of stock standard, dilute to volume, and mix. Ten ml. of the dilute 
standard contains 0.03 mg. 



H 2 



( 



Procedure : 

Into a 50 ml. volumetric flask place 15 ml. of -0.05% saponin solution. Add 1 ml. 
of blood. When laking is complete, add 10 ml. of 15% TCA. Dilute to the mark. Mix. 
Filter, Transfer 10 ml. of filtrate, 10 ml. of the dilute standard, and 10 ml. of 3% 
TCA respectively to three dry photometer tubes, and treat each as follows: Add 1 ml. 
of 0. 1% NaN0 2 solution. Mix. Let stand 3 mln. Add 1 ml. of 0. 5% ammonium sulfa- 
raate. Mix. Let stand 2 mln. Add 1 ml. of coupling reagent. Mix. Let stand 10 mln. 
Read in photometer. 













Mix, let 


Mix, let 


Mix, let stand 


Photo- 


TCA 








stand 3 


stand 3 


10 mln. Read 


meter 


blood 


on. 


3% 


0.1% 


mln. Add 


mln. Add 


In 


tube 


filtrate 


std. 


TCA 


NaNO z 


ammonium sulfa- 


coupling 


Photometer 


no. 


ml. 


ml. 


ml. 


ml. 


i .mate, ml. 


reag. , ml. 


T D 


1 


10 


10 


10 


1 

1 
1 


1 
1 
1 


1 
1 

1 


- 


2 




3 


100.0 0.000 



254 






Calculation: 



(D./DJ- 0.03(100/0. 2) 



(D U /D B )- 15 mg. of drug/100 ml. blood 



Comments: 

Saponin can be replaced by water if enough time is allowed for taking to become 
complete before addition of CCloCOOH. Some of the drug is present in an acetylated 
form. Determination of this form requires hydrolysis of the filtrate with acid before 
dlazotization and coupling. Some sulfa derivatives are so little soluble that it is advis- 
able to add 5 ml. of 2.5 N NaOH to the water in which the drug Is dlsolved to make the 
stock standard. Amount of color developed by equal weights of -sulfa drugs is approxi- 
mately inversely ae their molecular weight. It is better to use as a standard the par- 
ticular derivative which is being determined. 

For urine , use a 1 to 10 dilution with water. Saponin is not needed. Use the pro- 
cedure as for blood on this dilution including the treatment with TCA. 






255 



THIQCYANATE 

Co rum 






( 



Reference ; 

Bowler, II, G., Biochem. J. 38, 385 (1944). 

Principle ; 

A protein-free filtrate (trichloracetic acid) lo reacted with an excess of ferric Ions 
In 0.5 N_HH0 3 - which have been found to be optimum. The red color (ferric thlocyanate) 
is unstable efloaclally in sunlight. The color should be read immediately using 515 mu. 

Reagents ; 

1. Trichloracetic acid, 10% solution. Dissolve 10 grams in water, make to 100 ml. 
and filter. 

2. Ferric nitrate -nitric acid reagent. Dissolve 60 grams of ferric nitrate Fe(N0 3 ) 3 
9H 2 0, In 250 ml. of 2 N nitric acid, make to 500 ml. with water and filter. Concentra- 
ted nitric acid is approximately 16 N so that 2 N acid contains very nearly 31.5 ml. of 
cdncentrated acid in 250 ml. Keep in the dark. 

3. Standard thlocyanate solution. Prepare a standard solution containing 10 mg. SCN* 
per 100 ml. of solution, by diluting 1 to 100 with water a thlocyanate solution containing 

1 gram SCH per 100 ml. Standardize against silver nitrate solution. Twenty ml. of N/10 
silver nitrate would require 11.6 ml. of the thlocyanate solution. Or, If N/10 thlocya- 
nate Is kept In the laboratory, the standard thlocyanate (10 mg. per 100 ml.) can be pre- 
pared by diluting 1 ml. of this to 58 ml. with water. 

Procedure : 

Pipet 1.0 ml. of serum Into a teBt tube, add 6.5 ml. H 2 and 5.0 ml. 10% CClgCOOH. 
Mix and allow to stand 10 - 15 minutes. Filter through Whatman filter paper #40. 

To 5.0 ml, of filtrate add 5.0 ml. ferric nitrate reagent and mix well. (Keep out 
of bright light). 

Prepare a blank, using serum known not to contain thlocyanate and a standard as 
follows: 1 ml. normal serum / 6.5 ml. H„0 / 1.0 ml. thlocyanate standard and 5.0 ml. 
10% CCljCOOH. 



( 



Calculation: 



(D /D fl ) x 10 = m?. SCN/10Q ml. serum. 



Interpretation : 

Thiocyanates are used in the treatment of hypertension and in the estimation of 
total body water. Above about 12 mg. % toxic symptoms may appear. Therapeutic 
levels are about 10 mg. %. 



256 



THYMOL TURBIDITY 



Reference : 

Uucrga, J. de la. and Popper, H. , J. Lab. Clin. Med. , 34, 877 (1949). 
Rucci, II. , J. Lab. Clin. Med. , 32, 1266 (1947). 

Principle : 

It has been found that sera added to thymol solutions give turbidities, especially 
In liver disease such as infectious hepatitis. The amount of turbidity is measured against 
the standard turbidity tubes described under TURBIDITY STANDARDS, D. 329. 

Solutions : 

1. Thymol in alcohol 10% - 10 g. annydrous thymol dissolved up to 100 ml. with 95% 
ethyl alcohol. Keep In refrigerator. 

2. Half -strength Thymol-Buffer. Use only until turbid. 

Barbital (Barbituric acid) 2.76 g. 
Sodium Barbital 2. 06 g. 

Make up to 800 ml. in a 1 -liter volumetric flask, add 6.0 ml. of 10% alcoholic 
thymol solution, shake well to dissolve and fill with water to the mark. Mix. 

3. Full strength Thymol-Buffer (use only until turbid). 0.5 ml. of 10% thymol Is 
diluted to 100 ml. with half-strength Thymol-Buffer, shaking well to dissolve. 

Procedure : 

To 6.0 ml. of full strength Thymol-Buffer In a large (22 x 200 mm.) standardized 
photometer tube, add 0. 100 ml. of serum, using a ■to contain" plpet. Mix, allow to 
stand 30 minutes and read against Thymol-Buffer blank, at 644 mu. 

Calculation: 

From transmission reading, look up optical density and refer to graph of optical 
density vs. thymol or turbidity units. 

Example: 

A serum gave a T of 83.6 corresponding to sua optical density of 9. 078. This rep- 
resents 1.8 turbidity units as read from the graph. Normally, 9-4 units are obtained, 
(usually 1-2). Elevations up to 20 units are seen in liver disease, especially infectious 
hepatitis and cirrhosis. 

Precaution s: 

1. Clear, uncolored thymol buffer is essential. 

2. Good mixing of serum and buffer Is necessary. 

Notes and Interpretation: 

The change In and increase of gamma globulins are mainly responsible for positive 
results. Also, however, the amount of phospholipid, and qualitative changes In the al- 
bumin, are Important. 

257 



TIlANltAMINASE - Scrum 
(Scrum- glulamlc-oxnlacclic tranuam Inane ("S-GOT'7 



RcfcrcnccB: 






acid nucleotide (DPNHg) MDH acid 



As fast as oxalacetic acid Is formed It Is reduced to malic acid by the action of 
MDH, provided the concentrations of both MDH and DPNH. are high enough. Under 
the specified conditions of substrate concentration, pH and temperature, the rate of 
disappearance of DPNH, Is directly proportional to the S-GOT concentration. The 
reaction Is followed by observing the decrease In the characteristic light absorption 
peak of DPNH at 340 mu where DPN has virtually no absorption. 

This test Is highly specific for transaminase and Is reproducible with a standard 
deviation of L6 per cent. 






c 



1. Steinberg, D. , Baldwin, D. , and Oatrow, B. II. : A clinical method for the 
assay of scrum glutamic -oxalacetic transaminase. J. of Lab. and Clin. Med. 
48, 144-151 (1956). 

2. Cabaud, P., Lecper. R. , and Wroblewskt, F.: Color! metric measurement 
of scrum glutamic oxalacetic transaminase.' Am. J. of Clin. Path. 26:1101- 
1105 (1956). 

3. Wroblewskt, F. , Caband, P.: Colorlmetrlc measurement of serum glutamic- 
pyruvic transaminase. Am. J. Clin. Path. , 27, 235-9 (1957). 

Principle : 

Glutamlc-oxalacetic transaminase catalyzes the transfer of an amino group from 
aspartlc acid to alpha -ketoglutaric acid, yielding oxalacetic acid and glutamic acid, 
or the reverse: 

Aspartlc / alpha -ketoglutaric Oxalacetic / Glutamic 

acid acid S-GOTy acid acid 

This reaction Is coupled with the malic dehydrogenase (MDH) reaction: 

Oxalacetic / Reduced Dlphoaphopyrldine ^ Malic / DPN 



265 



Apparatus : 

This reaction can be followed in the Declcman DU npectrophotometer or the Bausch 
and Lomb'a spcctronlc 20 colorimeter (specially calibrated at 340 mu). Any spectro- 
photometer calibrated at 340 mu can be used. The lleckman DU spectrophotometer, 
however, affords somewhat greater precision. 

Reagents : 

1. Potassium phosphate buffer, 0.1M, pH7.4, 

13. 97 grams buffer quality anhydrous K„HP0 4 / 2.69 grams buffer quality 
anhydrous KHoPO, made up to 1 liter with water. Store in the cold. 

2. 0.2 M L-aspartic acid, sodium salt. 

2. 662 grams L-aspartic acid dissolved in 70 ml. of solution (1). Adjust to 
pH 7.4 (approximately 20 ml. 1 N NaOH). Dilute to 100 ml. Keep frozen. 

3. Reduced diphosphopyridine nucleotide (DPNH„). 

Available in different degrees of purity. If using 70% pureDPNH , take up 
10 mg. in 10 ml. of (1). If using 90/% pure DPNH 2 , take up 7.5 mg. in 10 ml. of (1). 
When diluted 1:10 as in the assay procedure this solution should give an optical d~n- 
sity about 0. 5 at 340 mu. 

4. Malic dehydrogenase (MDH). 

Potency varies from batch to batch. Enzyme should be diluted with (1) so 
as to contain 2000 MDH units/ml. Refrigerate. 

5. 0.1 M alpha-ketoglutarlc acid, sodium salt. 

1.47 g. alpha-ketoglutaric acid dissolved in 70 ml. of solution (1). Adjust 
to pH 7.4 (approximately 20 ml. 1 N^NaOH). Dilute to 100 ml. Keep frozen. 

Procedure : 

I. For each serum to be assayed prepare two tubes as follows: 



. 2 M aspartate 
MDH (2000 units /ml.) 
DPNH„ (0.75 to 1.0 mg./ml) 

Serum 

0. 1 M phosphate buffer 

2. After thoroughly mixing, allow tubes to stand for 30 minutes. This pre- 
incubation period is crucial to uxhaust the intrinsic DPNH 2 oxidizing capacity of 
the serum sample. 



259 



Control Tube 1 


Assay Tube 2 


0.5 ml. 


0.5 ml. 


0.1 ml. 


0.1ml. 


— 


0.3ml. 


0.3 ml. 


0.3 ml. 


1.9 ml. 


1,6 ml. 



3. After 30 minutes add 0.2 ml. of 0. 1 M. alpha-kctoglutarate to each tube and 
mix again. 

4. Using tube 1 as a blank, read the optical density of tube 2 at 340 mu exactly 
2, 4, 6, 10 and 15 minutes after step 3. For samples of low activity another read- 
ing can be taken at 20 minutes. With samples of high activity the optical density 
may fall below 0.200 In the first 6 minutes, indicating that the GOT level is probably 
over 100 units/ml. To obtain an accurate value the test should be repeated using 

a 1:2 or 1:4 dilution of the serum (diluted with solution 1). 






TEMPERATURE CONVERSION FACTORS 






Temperature 
°C 


f T 


Temperature 
°C 


f T 


21 


0.73 


30 


1.37 


22 


0.80 


31 


1.44 


23 


0.88 


32 


1.52 


24 


0.93 


33 


1.61 


25 


1.00 


34 


1.70 


26 


1.08 


35 


1.80 


27 


1.14 


36 


1.89 


28 


1.22 


37 


1.98 


29 


1.30 


38 


2.10 


Calculations-. 









One unit of GOT is the amount of enzyme that will cause the reading at 340 mu 
to decrease at the rate of 0.001 O.D. units per minute per centimeter light path at 
25°C. under the described conditions. 

Before any assay results are accepted for calculation, the linearity of the re- 
action rate is confirmed by inspection or plotting of the readings. Within the limits 
of error for reading the instrument, the rate of change In O. D. should remain con- 
stant during the assay. If the rate is linear, the difference between any two readings 
can be used in calculating results. Errors due to instrument instability, reading 
errors, etc. , are minimized by using the first and last readings. 









260 



GOT (unite/ml.) 



where: 



/o.Dj - O.D., 

* 4 - 4 



MDH blank*) x 1 

/ 7~ x 

*T ml. Borum 



1000 



O.D. . - optical denolty at t. 

X J- 

O.D.. - optical denolty at t„ 

< - time (minutes) 

f T = temperature conversion factor 

*MDH blank - see the Notes Section for significance. 

Sample Calculation : (A similar data sheet should be made out for all determinations) . 









Optical density 


readings 




Time (Mm) 


2 


4 


6 


10 


15 


Sample No. 1 


0.490 


0.482 


0.474 


0.457 


0.437 


Sample No. 2 


0.475 


0.470 


0.455 


0.427 


0.322 



"Sample No. 1' 



Temperature 



GOT (units/ml.) - 0.490 - 0.437 x 1_ x 

15-2 1.22 



1000 
0.3 



o 

28 C 






• 11.1 units/ ml. 






In calculating Sample No. 2 the optical density at t 1 would be 0.470 since it was 
at this point that the reaction began to fall at a constant rate. 

Standardization: 



A reference standard of the GOT enzyme is not at present available. However, 
if the following two criteria are met it can be concluded that the assay is working 
properly. 

1. The curve for DPNH„ disappearance Is strictly linear in all assays. 

2. The rate of the reaction Is directly proportional to GOT concentration. Graded 



261 



aliquota of the same scrum sample arc assayed on the same day. The rates of 
DPN1I 2 disappearance should be in direct proportion to the size of serum aliquot used. 

N OTES 

. 

MDH B lank. Preparations of malic dehydrogenase generally contain small but 
measurable amounts of GOT activity. The level of this GOT activity should be checked 
for each batch of MDH. The assay is run in the usual manner, substituting phosphate 
buffer for serum, and the observed rate of fall in optical density at 340 mu is sub- 
tracted from the observed rates in all assays. If the MDH blank is less than 0.0005 
per minute it can be neglected for most purposes. 

Temperature. As with all enzymatic reactions the rate of thlB coupled reaction 
varies markedly with temperature. For maximum accuracy and reproducibility, tubes 
should be incubated in a constant temperature bath. However, provided room tempera- 
ture is sufficiently constant, it is possible to obtain accurate and reproducible results 
Incubating the tubes in room air. 

Normal red blood cells contain GOT at a much higher concentration than normal 
serum. Hemolysis must be avoided during collection of GOT concentrations In serum 
and plasma. Heparin and oxalate do not interfere with the assay. GOT in serum Is 
stable at refrigerator temperatures for 3 weeks. 

Glutamic -oxalacetlc transaminase is only one of many transaminases. Other 
transaminases can be determined by this method by using the appropriate substrate 
and enzyme. For example glutamic-pyruvic transaminase can be determined by this 
procedure by substituting alanine for aspartlc acid and lactic dehydrogenase for malic 
dehydrogenase. 

When using a Beckman DU spectrophotometer it is not necessary to run a separ- 
ate blank for each serum to be assayed. It is possible to assay three samples at a time 
by using a common blank (this common blank can be prepared by diluting serum with 
phosphate buffer In the ratio that it is diluted In the assay samples). Satisfactory re- 
sults can be obtained by this modification. 

It has been reported that aspirin administration can influence the serum activity 
of glutamic -oxalacetlc and glutamic -pyruvic transaminases In children (Manso, C, 
A. Toranta, and I. Nydlc. Effect of aspirin administration on serum glutamic -oxala- 
cetlc and glutamic -pyruvic tansamlnases In children. Proc. Soc. Exp. Biol. & Med. 
93: 84-88 (1956), 

Interpretation: 

Normal values are usually be* ween 10 and 33 unlts/mt. of serum for glutamic - 
oxalacetlc transaminase. GOT levels rise within 24 hours and fall to normal by the 



262 



6(li day after acute myocardial Infarction. In recent myocardial Infarction the values 
are elevated often to 100-200 units/ml. and occasionally to several thousand units. 
Elevations are also seen In Jaundiced paMenttj with active liver disease. 

The measurement of serum glutamic -pyruvic transaminase alterations has been 
found to be a useful tool in the diagnosis and study of acute hepatic disease and appears 
to be more sensitive than S-GQT In depicting acute hepatocellular damage. Normal 
values for glutamic-pyruvic transaminase arc usually between 6 and 35 units/ml. of 
serum. 









263 



UREA NITROGEN 
Blood and Urine 

Refe rence : 

Boutwell, J.H. , Jr. ; Clin. Cliein. , 3, 205 (1S>57). 

Print- iT ;Ie: 

A dilution of blood (or urine) in water Is Incubated with urea&e to convert urea to 
ammonia . 

(NH z ) 2 CO / H 2 > 2KH 3 / COg 

A protein free filtrate is then prepared. A portion Ib neEBlerized, and Is compared 
photometrically with a standard urea solution treated similarly, using 515 mu wave- 
length. 

Reagents ; 

1. Ethylenediaminetetraacetlc acid (EDTA). (Trade names, VerBene, SequeEtrene), 
1%, pH 6. 8. Weigh out 1.0 g. of ethylenediaminetetraacetlc acid, suspend in about 

50 ml. of distilled H„0, neutralize with KaOII to pH 6,8 (nitrazfne paper or glass elec- 
trode) and dilute up to 100 ml. 

2. Urease solution, 0.5%. Weigh out 0.50 g. of crude commercial urease (Sigma 
#2) and add to 100 ml. of the neutralized EDTA. Mix to dissolve. Keep refrigerated. 
This solution maintains its original activity for at least 4-6 weeks in the cold. Jack 
bean meal extract may be used. See notes below. 

3. Acetate buffer. Dissolve 100 g. NaC 2 H 3 2 '3H 2 and 1. 1 ml. glacial acetic 
acid in water, dilute to 500 ml. and mix. 

4. Urease-buffer mixture. Add 2 ml. urease solution and 2 rol. acetate buffer to 
6 ml. HoO and mix. Prepare on day of use. 

5. Zinc sulfate, 1%; Ba(OH) 2> 0.06 N. Seep. 76, Coprotelni-zlu^ Methods. 

6. Standard urea solution, stock. Dissolve 0.4287 g. of pure, dry urea In water 
and dilute to 1 liter; mix. Add2 drops toluene. This solution contains 20 mg. of urea 
nitrogenjper 100 ml. 

7. Urea standard, dilute. Dilute 5.000 ml, of stock standard urea solution to 100 
ml. Willi water. Mix, Five ml. contains 0.05 mg. urea nitrogen. 

8. Ammonium sulfate, stock standard. Weigh out 0.9434 ammonium Bulfate (pure 
and dry), dissolve In water, and add one drop of concentrated H2SO4, dilute to 1 liter 
with water. Mix. Five ml. contain 20 mg, ammonia nitrogen per 100 ml. 



264 



( 



9. Ammonium sulfate, dilute standard. Dilute 5 ml. of stock standard ammonium 
sulfate to 100 ml. with water mix. Five ml. contains 0.05 mg. of nitrogen. 

10. Nessler's Stock Solution (Koch-McMeekin). Into a 500 ml. Florence flask place 
22.5 g. of Iodine, 30 g. of KI, 20 ml. of water, and 30 g. of pure metallic mercury. 
Shake the mixture by rotation. The iodine and iodide will quickly dissolve, and the 
flask and contents will become hot. In a short while the red iodine color will begin to 
fade. Before all red color disappears cool rapidly under cold water, and when the solu- 
tion has only a email amount of red color, decant the aqueous solution carefully from 
.the excess mercury, which. should be saved and recovered. Transfer all of the aqueous 

portion to a cylinder, and dilute up to 200 ml. with water. 

11. Iodinated Nessler's Solution. To 100 ml. of Nessler's Stock Solution add 1.0 g. 
of pure L, (solid). Mix. Allow to stand until dissolved, mixing occasionally. This solu- 
tion will require several days. If necessary, the solution may be used before the Iodine 
is completely dissolved. 

12. NaOH, 2.6 N. Dilute 139 ml. of 18 N NaOH (see Solutions p. 30) up to 1 liter 
with water. Standardize and adjust normality to a figure between 2. 48 N and 2. 52 N. 

Procedure : 

1. To 5.00 ml. distilled water In a test tube, add (by 8 times rinsing), 0.ZU0 ml. 
biood from a pipet calibrated "to contain. " Mix. 

2. In addition to the blood tube, prepare similar tubes containing: 

a. 5 ml. H„0 

b. 5 ml. dilute urea standard 

c. 5 ml. dilute ammonium sulfate standard (if desired) 

d. 1 - 5 ml. of a 1:200 dilution of urine (the exact amount depends pn the urine 
urea concentration) made up to 5 ml. with water. 

3. To each tube add 1.0 ml. urease buffer mixture. Mix. Incubate in water bath 
at 50° C. for 10 minutes. 

4. Add to each tube 2.00 ml. of zinc sulfate, 1%. Mix. 

5. Immerse tubes in a boiling water bath for c -^nutes. Remove and cool. 

6. Add 2.0 ml. Ba(OH) 2 , 0.06 N. Mix >; vigorous shaking. Centrifuge. 

7. Transfer 5 ml. allquots (by dv^s of cotton tipped pipet) to matched photometer 
tubes. 

8. To each tube add 1. ml. ^dinated Nessler's Solution. Mix. Then add rapidly 
(blow In) 1.0 ml. 2.5 N NaOH, mix each tube immediately after alkalinUing. 

265 



». Allow to aland 5 minutes, read within an hour against the reagent blank set at 
100. 

L0. If the tranemltiance Is above 90 or below 10, repeat, with more or less sample 
or filtrate. 

Calculations : 

A. Blood. C u = <V^>* C s x ^j' or C u = <VD B )x 5(0.06/10) 5 x ™° 2/lQ2 

or C„ = (D U /DJ x 25.6 = m K* urea nitrogen/100 ml. 

B. Urine. Using 2 ml. of 1:200 dilution, 

C„ , ,D U /D S ,,5X(C.05AO,. W ^L_ ) . OVD.,.5. V (oU1 . 

mg. urea nitrogen/volume 
Comments: 

1. NaOH, 0.06 N, may be used Instead of Ba(OH)-, with only minute and occasion- 
al appearance of turbidities in the final solution. 

2. Addition of iodlnated Nessler's solutioirand NaOH 2.5N should be carried out 
at room temperature. 

3. Urease is protected from heavy metal poisons by EDTA, by blood proteins and 
by some other substances, but in urine dilutions and in pure urea solutions, not enough 
of this protection Is present. Hence, care should be taken to separate tubes for Neasl- 
erlzation (mercury containing) from those used for incubation. Urease poisoning ac- 
counts for the occasional apparent failure of urease to act. 

4. The use of Iodlnated Nessler's solution prevents the rea< tlon of such reducing 
substance^ as creatinine and url' acid with the alkaline Nessler's reagent. Other oxidiz- 
ing agents have a similar effect. 

5. The heating of the solutions In the presence of ZnSO. is necessary to insure com- 
plete removal of protein by the ZnSO^-Ba(OH> 2 reagents. 

6. Reproducibility of color depends on rapid mixing when the alkali Is aaded (Step 8). 

7. Obviously, the sum of urine urea nitrogen and urine ammonia is determined by 
this procedure. If a correction for urine ammonia Is desired, add the ZnSO, before the 
urease, heat in the boiling water bath Immediately, cool and proceed as above, using 
the value obtained as a blank value to be subtracted from the urea value. 

266 



( 



( 



8. If greater pliotometric accuracy in the low normal range is desired, use 5.0 ml. 
of a Foltn-Wu 1:10 blood filtrate (instead of the 5.0 ml. of water / 0. 2 ml. blood). This 
then will result In a 0. 25 ml. aliquot and C u = <D U /D B ) ■ 10 mg. % urea N In blood. 



9. Urine Ammonia - Proceed as above with the following- changes: 

a. Use a 1:100 dilution of urine. 

b. Omit the addition of urease - buffer. Step 3. 

c. Omit the urea standard; use the ammonium sulfate standard. 

Calculation: Using 5.00 ml. of 1:100 dilution 

C u = (Dy/Dg) - 100 = mg. NH 3 -Nper 100 ml. brine 

10 . Jack bean meal extract may be prepared as follows: 

Urease extract . Weigh out 1.0 g. of ethylenedlamlnetetraacetlc acid (EOT A, 
Versene, Sequestrene) , suspend It in about 50 ml. of distilled water, and 
add 10% NaOH with stirring until the EDTA goes Into solution and a pH of 
6.8 1b reached. In this solution dissolve 20 g. of Htf^HgOg* SSJO and 
0.22 ml. of glacial acetic acid, dilute to 100 ml. with water, and mix. 
Shake this solution, with 15 g. of jack bean meal for 16 minutes. Centrifuge 
at 2000 rpm for 2 minutes, and decant all liquid portion that will poor off. 
Addition of toluene and refrigeration will preserve the extract for several 
weeks. Mix before using. This extract has a pH around 6. 26 at room tem- 
perature. 

Acetate buffer solution. Suspend 10 g. of EDTA in about 600 ml. of water, 
dissolve with NaOH and bring to pH 6. 8. Add 50 g. of NaCjHjOj- 3HjO and 
0.55 ml. of glacial acetic add. dissolve, dilute to 1 liter, and mix. 

Urease -buffer mixture . Add 1 ml. of urease extract to 60 ml. of acetate 
buffer solution. Stir up before use. Make fresh daily. 



267 



UREA 
Blood and Urine 

References : 

1. Karr, W.G. : J. Lab. and Clin. Med. 9, 329 (1924). 

2. Connerty, H.V. , BrJggs, A,R. and Eaton, E.H. , Jr., Am. J; Clin. Path., 
25, 1321 (1955). 

Principle : 

A tungstic acid filtrate is treated with ureaee (using a pyrophosphate buffer) to con- 
vert urea to ammonia. The ammonia is then reacted with Nessler's reagent to produce 
a colloidal yellow color which is compared to the color produced by a standard urea 
solution treated similarly. 

CO(NH 2 ) 2 / H 2 Q (urease)^ co ^ j 2 NH 
(Urea) (ammonia) 

Reagents : 

1. Deproteinizing reagents, seep. 76. 

2. Pyrophosphate buffer. Dissolve 14 g. sodium pyrophosphate (Na.PnOir ■ lOHoO) 
and 2. ml. phosphoric acid (HgP0 4 , 85%) in distilled water and dilute to 100 ml. 

3. Urease solution. 

Into a 200 ml. flask, place 100 ml. of about 30% ethyl alcohol. Add 30 g. Jack 
bean meal and 1 ml. pyrophosphate buffer. Stopper and shake vigorously for five min- 
utes. Centrifuge for 1/2 hour In 12 ml. heavy wall centrifuge tubes, covering the 
mouth of the tube with aluminum foil. Transfer the supernatant to a glass -stoppered 
bottle and store under refrigeration. 

4. Potassium mercuric Iodide solution: 

Into a 500-ec. Florence flask place 150 gm. of potassium iodide (KI) A.C.S. , 
100 gm. of elemental iodine, A.C.8. , and 100 ml. of distilled water. Shake the contents 
of the flask until the iodine has completely dissolved. Add 150 gm. of metallic mer- 
cury, A.C.S. Shake the flask continuously and vigorously for 7 to 15 minutes until the 
dark red color of the iodine has become visibly lighter though still red. The solution be- 
comes quite hot at this stage. After the solution becomes pale though still red, cool the 
flask under a running water tap and continue shaking until the reddish color of iodine has 
been replaced by the greenish-yellow color of the double iodide. If the solution is prop- 
erly prepared, it is clear and there is a residue of shining metallic mercury. Decant the 
solution in a 2-liter volumetric flask, washing the mercury and flask with copious quanti- 
ties of distilled water. Dilute the solution and washings to 2 liters. If the cooling was 
begun in time, the resulting reagent Is clear enough for Immediate dilution with 10 per 
cent sodium hydroxide and water for preparation of Nessler's solution. Never discard 
mercury by pouring it into a sink because itj will alloy with the lead of plumbing fix- 
tures and later cause leaks, 

5. Nessler' s solution 

Into a 2-liter volumetric flask place 300 ml. of potassium mercuric iodide 



268 






( 



solution and 1,400 ml. of 10 per cent sodium hydroxide. Dilute to the mark with dis- 
tilled water and mix thoroughly. Allow the solution to stand several days before use 
to allow the yellow precipitate to settle. 

6. Stock urea standard : 

Dissolve 0. 1286 gm. of urea, weighed on an analytical balance. In distilled 
water and dilute to 200 ml. in a volumetric flask. 

7. Working urea standard : 

Transfer 5 ml. of stock urea standard with a volumetric plpet to a 100-ml. 
flask and make up to the mark with distilled water. 1 ml. of urea standard contains 
0.015 mg. of nitrogen. 

8. L,'KI solution 

Add 2 g. Lj to 3 g. H, dissolve In a little water and dilute to 100 ml. 

Procedure : 

1. Prepare a 1:10 tungstic acid filtrate of whole blood, see p. 70. 

2. Into a clean, dry test tube calibrated at 9 ml. , place 1 ml. of the protein-free 
filtrate. 

3. Into similar test tubes, place 1 ml. of water (for a blank) and 1 ml. of urea 
standard containing 0.015 urea nitrogen. 

4. To each tube add 1 drop of pyrophosphate buffer and 1 drop of urease solution. 
Mix well. 

5. Incubate at 60°C (t 6°C.) for 10 minutes. Cool. Add 1 drop of 2% I*KI solu- 
tion. 

0. Dilute each tube to exactly 0.0 ml. Mix well. 

7. Cool to below 20°C. in a water bath or refrigerator. 

8. Add to each tube in turn 1.0 ml. alkaline Nessler's solution, mixing Immediate- 
ly after each addition. 

0. Allow to stand for 5 minutes, read against the reagent blank within 30 minutes 
using a wavelength of 500 mu (or a green filter). 

Calculation : 

(Du/Ds) x C x !5£ * mg. urea If per 100 ml. blood 
fl 0.1 

(Du/Ds) x 0.015 x 1000 = mg. urea Nper 100 ml. blood 

or 
(Du/Ds) x 16 = mg. %urea N 

Urine Urea: 



1. Dilute urine 1 to 500 with water. 

2. Proceed as outlined above, Steps 2-9. 



269 



UREA 

Visual Colorlmelric Determination 



Urine Urea : 

Prepare a 1:800 dilution and proceed as above. 









Interp retation: 

Normal range: 8-18 mg. urea N per 100 ml. blood 
(equivalent to 20-36 mg. urea) 

Increased values are seen in renal failure, urinary obstruction, and oliguria. la 
acute nephritis, values as high as 200 mg. % may be reached. Decreased values are 
seen in acute hepatic failure and in normal pregnancy. 






270 






Principle : 

Since the yellow Nesslers- ammonia complex color Is difficult to measure by visual 
colortmetry, It Is necessary to work with, more concentrated solutions. 

P rocedure : 

1. Into a 25 x 200 mm. test tube calibrated at 20 ml. , place 6.0 ml. of a protein- 
free filtrate of whole blood. 

2. Into similar teat tubes place 6.0 ml. of a urea standard containing 0.076 mg. 
urea nitrogen. 

5. Add 6 drops of pyrophosphate buffer and 6 drops of urease solution. Mix well. 
4. Incubate at 60°C (£ 6°C.) for 10 minutes. Cool. 

6. Add 2 drops 2% Ig- KI. Dilute to 20.0 ml. Mix well. Cool to below SQ°C. 

6. Add with Immediate rapid mixing 2.0 ml. alkaline Nessler's solution, 

7 . Allow to stand 6 minutes and compare against the standard in a visual colori- 
meter, if possible a blue or green light source should be used. 

Calculation 

_JL C H 129. = mg. urea N per 10V mi. whole blood 
B u ° 0.6 

(R./1U 0.076 15i - 

(Rg/Ry) x 15 s mg.ftureaN 



(. 



URIC ACID 
Serum and Urine 

Reference : 

Brown, H. . J. Btol. Chem. 158, 601, (1945). 

Buchanan, O. H. , Christ man, A. A. and Block, W. T. , J. Btol. Chem. 167, 
181, (1945). 

Hermann, F. , and Kiksteln, J. Biol, Chem. , 211, (1) 149. (1954). 

Principle: 

This method, like most, depends upon the great ease with which uric acid is oxl- 
di?prl in either acid or alkaline media. The oxidation la carried out by a special phos- 
photungstlc acid In the presence of alkali and sodium cyanide (to Intensify the color 
obtained) . 

Reagents : 

1. Uric Acid Reagent (Phosphotungstlc Acid): Into a 1000 ml. round bottom flask, 
which has been cleaned with dlchromate sulfuric acid cleaning solution, place 150 ml. 
water, 100 g. Na^WO^HjO (Folln). 16.3 ml. of 65% (13.8 M) H 3 P0 4 and 16.6 ml. 
cone. HgSC^ (18 M .) Mix. Boil gently, two hours, using a reflux condenser or funnel- 
flask combination. Cool, and dilute to one liter with water. The reagent is stable In- 
definitely. 

2. Sodium Cyanide - Sodium Carbonate. Dissolve 60 g. NaCN and 20 g. Na^CO, 
(anhydrous) dilute to one liter with water. Keep refrigerated. 

3. Urea - 50%. Dissolve 50 g. reagent grade urea up to 100 ml. with water. 

4. Urea-Cyanide. Just before use mix equal volumes of reagents (2) and (3). Dis- 
card excess each day. Do not keep. 

5. Uric Add Stock Standard. Place 1.000 g. uric acid In a funnel on a 300 ml. 
Erlenmeyer flask. Place about 0.45 - 0. 50 g. lithium carbonate In a beaker In about 
lGQ ml. of water and beat at 60°C. and stir until all Is dissolved. Rinse the uric acid 
into the flask with this hot carbonate solution and shake until the uric acid is dissolved, 
cool under running water and transfer to a one-liter volumetric flask. Rinse and dilute 
to a volume of 400-500 ml. Add 26 ml. of 40% formaldehyde and after thorough mixing 
add 3 ml. of glacial acetic acid and dilute to mark, Keep In a tightly stoppered bottle 
(brown) in the dark, 1 ml. - 1 mg. 

6. Uric Acid Standard Dilute. Transfer 1.000 ml. of uric acid stock standard to 
250 ml. In a volumetric flask, dilute to mark with water. Make fresh each day. 1 ml. 
0.004 mg. 

Procedure ; 

A. Deprotelnlzation: The usual 1:10 tuugstlc acid filtrate of serum (Protein-free Fil- 
trates, p. 76 may be used ) . 

B. Color development: To a photometer cuvet add 1 ml. H_0, 1 ml. serum filtrate 
and 4 ml. urea-cyanide mixture, mix well. Then add 1 ml. uric acid reagent, and 
Immediately mix well. 



271 



Set up a blank tube (2 ml. H 2 0>; Sj tube {1 ml. H„0 / 1 ml. dilute standard); and 
8 2 tube (2 ml. dilute standard) and develop color as above. 

Allow all tubes to stand at room temperature for 00 minutes and compare photo- 
metrically (640 mu). 



Calculation; 



(D u /D s ) C fl M_ = C u (mg. per 100 ml.) 



For S i' AW 0.004 x -^1 = P U /D B ) x 4 = mg. % uxlc acid 



Urine: 



A dilution of urine (1:100 usually la sufficient) la made and treated as blood filtrate. 

Notes : 

Sodium cyanide purity le essential to the success of the determination. Impure 
cyanide gives Insufficient color, Increases instability of the color, and results In the 
development of a yellow or brown color upon standing. Purification can be made as 
follows: (Medical and Public Health Laboratory Methods, Simmons, J.T. , Gentzkow, 
C. J. , Lea tr, Feblger, 1955). m a good hood, add 225 g. NaCN (reagent grade pre- 
viously found to be unsatisfactory) to a 600 ml. flask. Add 200 ml. boiling water. Stir 
a few minutes. Filter through filter paper using vacuum. Cover. Allow to cool over- 
night in the refrigerator. Collect crystals using vacuum filter. Wash twice with 20 ml. 
Ice water. Partially dry, store moist In a brown Jar. Substances other than uric acid 
influence the amount of color obtained, so the method is not entirely specific. Also 
some of the uric acid is lost by deproteinizatlon. Another difficulty, eliminated In the 
procedure given, was the fact that Beer's Law was not obeyed, In the present proce- 
dure Beer's Law Is obeyed up to 0.01 mg. uric acid in the tube, and this Is represented 
by an optical density of about 0.730, and corresponds to 10 mg. % uric acid. 






272 



/ilNC TURBIDITY (gatnmu globulin) 
Serum 

Reference : 

Kunkel. Henry G. , Proc. Soc. Exp. Biol. & Med. , 66, 217-224 (1947). 

Principle : 

The gamma glubulln is precipitated by zinc sulfate at a pH of 7.5 (barbiturate 
buffer). The turbid solution is measured photometrically, at 644 mil, using the 
standard turbidities of Shank and Hoaglund (see Turbidity Standards, p. 329. 
but expressed In MacLaglan units. 

Reagents : 



Mix the following reagents: 
ZnS0 4 -7H 2 
Barbituric acid 
Sodium barbiturate 

H 2° 


0. 024 g. (analytical balance) 

0.280 g. 

0.210 g. 

To make 100 ml. 


Procedure: 





1. Place 6.0 ml. of the zinc reagent in a photometer tube. 

2. Add with a micro-pipet 0. 100 ml. of serum, rinsing out 6-6 times. 

3. Mix well. Allow to stand 30 minutes. 

4 . Shake . Read in photometer against a reagent blank, using 644 mu. 
Calculation : 

1. Look up optical density (TvsD table. Appendix) 

2. Refer to calibration chart of turbidity for units of turbidity p. 329. 

Interpretation: 

The normal range lies between 2 and 8 units of turbidity. Certain sera with globu- 
lin levels above 6 g. % may show values as high as 80 units. The reaction is not specific 
for liver disease. The test was advocated by the author as being of particular value 
when applied serially throughout the course of acute Illness. He found the sine 
turbidity teat to be the moat sensitive method of detecting a lingering hepatitis. He 
also used It to follow development of antibodies after scarlet fever convalescence and 
In developing rheumatic: fever. 

273 



TOXICOLOGY 



From the history of ancient medicine, records and traditions Indicate that •-■v.Jti 
before the development of modern scientific methods there was In the field of medicine 
a knowledge of the action of venom poisons upon various animals. Although the med- 
ical profession" of the early ancients consisted of superstitious beliefs, It was due to 
this very practice that there developed a school of anatomical observations. This was 
the observation of the entrails of animals. The action of poison was, therefore, ob- 
served. 

Ancient literature, also, bears many allusions to the knowledge of poisons. Ovid, 
the poet, for example, between the years 43 B.C. and 17 A.D. , wrote that the arrows 
of Hercules were charged with the venoms of the Lernetan serpent. It Is from this 
method of poisoning that the word poison (Latin - toxlcum for poisoning, originally 
arrow poisoning is derived (I). 

Toxicology, therefore, Is the science of poisons, their origin, their properties, 
their action on living tissue, the treatment to counteract their harmful effects, and 
the methods of their detection by chemical or other means (2). 

In order to understand the true meaning of toxicology a definition is needed for a 
poison, which may be thought of as any substance which when Introduced Into the living 
body and acting chemically, is capable of causing serious disturbance of body functions 
or death of an ordinary Individual with average health (3). For our purposes In the 
Navy, only the poisons which need be present in relatively small quantity in order to 
cause Illness or death are of significance. 

Poisons originate from animal, vegetable, and mineral sources as well as by syn- 
thetic means. The properties of poisons may be acidic, basic, or neutral; organic or 
Inorganic . 

The action of poisons on the system disturbs the normal functioning of a vital body 
process. The common symptoms may vary from vomiting, convulsions, coma, deliri- 
um, paralysis, dilation or constriction of the pupils, rapid or slow respiration, to 
miscellaneous signs such as blood changes, disturbed vision, urinary changes, abnor- 
mal coloring of the skin, and characteristic odors on the breath. 

The minimum lethal dose of a poison is the smallest quantity of the substance which 
has proven fatal. It varies considerably with each Individual poison, and each poison 
differs from itself according to the mode of administration. Also certain individuals 
have idiosyncrasies which account for further variations of the action on the body. 
There are many factors which contribute to tide widespread action. 



( 



274 



The form In which a solid, liquid, or gaseous material may be taken varies consider- 
ably; particle size and solubilities being Instrumental In affecting toxicity. The poison 
may also enter the body in a variety of ways. The routes of administration in order of 
speed of absorption are intravenous. Inhalation, intramuscular, rectal, vaginal, and 
oral. The oral route being last because of slow absorption from the stomach. Also 
poisons are often partially eliminated by vomiting, and protein foods in the stomach 
act in some cases as an antidote for metallic poisons. 

The minimum lethal dose of a poison for an average 160 pound man varies from 
10 mg. aconltlne, 60 mg. nicotine, 100 mg. strychnine, 200 mg. arsenic, 1 to 6 gm. 
barbiturate, 15 gm. D.D.T. , to one quart of whiskey (4). 

The onset of illness is usually sudden, but not always. Symptoms usually but not 
necessarily follow the taking of food, drink, or medicine. The usual steps of treat - 
ment t aken are: 

a. The removal of the poison from the body. 

b. The administration of antidotes. 

c. Supportive or symptomatic therapy. 

In the removal of poison from the body there are two areas of consideration - one 
Is the artificial expulsion and the other is natural elimination. Toxic substances can 
be removed by the use of the stomach tube, unless corrosives or strychnine are sus- 
pected. An emetic is very useful and easily obtainable. The chief avenues of natural 
elimination are via the kidneys, lungs, skin, and Intestines. 

In considering the second step in treatment, an antidote Is any agent which will 
remove or prevent the absorption of a poison, or change its toxic properties, or 
counteract its physiologic effects. Antidotes act mechanically by removing the poison 
or by preventing its absorption by enveloping the poison in some insoluble material. 
They act chemically by changing the composition of the poison so that an Insoluble 
compound Is formed or by oxidizing the poison to nontoxic constituents. A physiologic 
antidote Is one whose physiologic action on the body is opposite to that of the poison. 

Symptomatic treatment, the third step, consists of cardiovascular, respiratory, 
or cortical stimulation and keeping the patient quiet and warm to combat collapse or 
shock. 

The occurrence of poisoning may be accidental, suicidal, or criminal. Fortu- 
nately we do not encounter poisonings to any great extent of the latter type In the Navy. 
The rapid strides made in the science of toxicology have largely discouraged poisoning 
as a form of criminal attack. As suicidal agents, barbiturates and carbon monoxide 
are two that are often employed. 



275 



As a rule, the liver is the organ which detoxifies most of the poisons held In the 
system. The lungs are the principal organ of excretion of poisons producing their 
effects by Inhalation. 

The purpose of the Toxicology Laboratory at the Naval Medical School, National 
Naval Medical Center, Is to detect qualitatively and quantitatively toxic substances In 
organic and Inorganic material. The detection of poisons In tissue Is an Intricate pro- 
cedure which demands specialized training. Occasionally a poison can be detected by 
gross examination of various parts of the body. For example the heavy metals are ex- 
creted In the saliva and produce characteristic effects on the mouth and lips. However, 
chemical tests are the primary means of detection. 

References 

1. McNally, W.D. , Toxicology, Industrial Medicine, Chicago, p. 1, 1937. 

2. Webster, W. W. , Legal Medicine and Toxicology, W. B. Saunders Company, 
p. 318, 1930. 

3. Goodman, L. and Oilman, A. . The Pharmacological Basis of Therapeutics, The 
MacMlllan Company, New York, p. 23, 1941. 



4. Kaye, 8, , Handbook of Emergency Toxicology, Charles C. Thomas, pp. 1-24, 1954. 
B. Bamford, F. , Poisons, Third Edition, The Blakleton Co. , Philadelphia, 1961. 
6. Oradwohl, Legal Medicine, Mosby, p. 699, 1954. 
T. Thlenes, L. H. , and Haley. T. J. , Clinical Toxicology, Lea and Fablger, 1949. 



( 



276 



REINSCfl TEST FOR ARSENIC, MERCURY, BISMUTH, SILVER, 
AND ANTIMONY, USING MINCED TISSUE , BLOOD OR URINE 

Reference : 

J. F. Pract: Chem. Leipa. 24, 244 (1841) 

Principle: 

The common metal poisons combine with or form a deposit on copper in an 
acid media. 

Apparatus : 

No special apparatus needed. 

Reagents: 

1, 5 per cent HC1 solution. 

2. Arsenic-free copper foil strips (1/8" x 3/4") or 18 gage wire (coiled 
1/4" x 1/2"). 

Procedure: 

1. Place 5-10 g. of material in a suitable beaker or flask. 

2. Add 25 to 35 ml. of 5% HC1 solution. 

3. Add one piece of Cu foil or wire which has been cleaned with concentrated HCt 
until bright and shiny. (Immerse strip In acid for 1-2 rain. ) 

4. Cover flask or beaker with watchglass to decrease evaporation. 

5. Place flask on hotplate and bring to boil. 

6. Check from time to time to notice any change In color of the copper. 

7. Add 5% HC1 solution as needed to replenish loss due to evaporation. 

8. Boil for 45 minutes. 

9. Remove Cu, wash with water, alcohol, and ether, then examine. 

10. Silvery gray indicates tig t Bhlny gray Ag, metallic blue-black Bi, and black 
either As or Sb. 

11. A reagent blank is run for comparison. 

Calculations : None. 

Standardization or Calibration: None. 



Notes: 



1. Do not handle Cu with fingers. Use forceps. 

277 



2. The 1ICI concentration In the lest must be from 2 to 8 per cent. If less than 
2 per cent certain arsenic compounds will not deposit. If more than 8 per cent the 
arsenic may be lost as arsine gas. 



3. The deposit is not formed In the presence of oxidizing agents. 






4. Compounds also causing stains on the Cu include sulfur (most common), sel- 
enium, gold, platinum, and palladium. 

Interpretation : 

1. This test is significant only when negative. 

2. A positive test must be confirmed by other tests. 

3. Sensitivity of tests as follows: 

As 1 mlcrogram/gm or ml 



Bl 5 

Hg 25 

Sb 5 

Ag 10 



ir it •• 

•t ii H 

it n ti 

it tt ti 



( 






278 



ARSENIC DETERMINATION IN ORGANIC MATERIAL 



References : 

1. Association of Official Agricultural Chemists, 7th Ed. , p. 369 (1950) 

2. Sandell, E.B.: Ind. & Eng. Chem. (Anal. Ed.) U, 82 (1942) 

3. Bamford, F.: Poisons, The Flakiston Co. , 3rd Ed. , pp. 76-77 (1951) 

4. Glaister, J.: Medical Jurisprudence and Toxicology, E. 6 S; Livingstone, Ltd., 
9th Ed. , p. 545-547 (1*50) 

Principle : 

Organic material is destroyed by dry ashing. The arsenic (and Sb) are rendered 
nonvolatile by forming the stable magnesium pyroarsenate (and antimonate). The dry 
ash is dissolved in hydrochloric acid and introduced into a Gutzeit apparatus where the 
liberated arsine is collected on glass wool impregnated with mercuric bromide. B 
arsine is present, a yellow to dark brown stain will form which constitutes a qualitative 
test. If positive, the arsenic will then be subjected to a quantitative teBt by being con- 
verted to molybdenum blue and compared photometrically. 

Apparatus : 

1. Photoelectric colorimeter. 

2. Gutzeit apparatus - the collection tube is packed in the lower end with absorbent 
cotton dampened with lead acetate solution. Blow gently thru tube to make sure gas 
can pass freely. The glass wool is inserted thru the upper-most end to about 1 1/2" 
from the bottom of the tube. It should not be tightly packed but should allow free passage 
of gases. 

Reagents : 

1. Stannous chloride solution - Dissolve 40 g. As-free SnC^" 2H 2 in As-free 
cone. HC1 and dilute to 100 ml. with same strength MCI. 

2. Potassium Iodide solution - 15 g. As-free KI in water and dilute to 100 mt. 

3. Lead Acetate solution - dissolve 10 g. of lead acetate Pb(Ac) 2 SH^O In water 
and dilute to 100 ml. 

4. Zinc - 20-30 mesh - As-free. 

5. Glass wool - pyrex brand. Wa3h in 10% NaOH and then in dilute HN0 3 (hot). 
Rinse with triple distilled water and dry. The wool is then Impregnated by soaking in 
5% mercuric bromide solution in 95% ethanol. Excess solution la pressed out between 
paper towels and allowed to air-dry. 

6. 6 N sulfuric acid (approx.):* Dilute 16.7 ml. concentrated H 2 S0 4 acid to 
100 ml. in distilled water. 

7. KMn0 4 solution: Dissolve 0. 1 g. of KMnO^ in water and dilute to 100 ml. 
Note: prepare fresh daily, 



279 



8. Ammonium molybdate solution - Dltiaolve 1 g. of ammonium molybdate la 
10 ml. of water and M nil. of 6 N sulfuric acid. 

i). Hydrazine sulfate solution - Dissolve 0. 15 g. of hydrazine sulfate in 100 ml. 
of water. Note : prepare fresh daily. 

10. COLOK REAGENT: Mix 5 ml. of ammonium molybdate solution and 5 ml, of 
hydrazine sulfate solution and dilute to 50 ml. 

11. Standard Arsenic solution - Dissolve 1. 32 g. of Bureau of Standards As 2 3 In 
25 ml. of 20% NaOH, saturate with C0 2> and dilute to 1 liter with fresh triple distilled 
water. 1 ml. equals 1 milligram of arsenic. Prepare fresh daily a dilute standard by 
taking 1 ml. of the above standard and diluting to 1 liter with water. 1 ml. equals 

1 microgram (gamma). 

12. Magnesium nitrate solution - Dissolve 200 g. of reagent magnesium nitrate in 
160 ml. of water. 

13. HC1 - Approximately 22% As-free HC1. 

14. MgO - Reagent grade powder. 

Procedure : 

1. Weigh 10-15 g. of organic material into a suitable Vycor or silica dish (not 
suitable for bone). 

2. Add 4 ml. of Mg(NOg)2 solution for every 10 g. of tissue taken and sufficient 
MgO powder to render the mixture alkaline. (2g. MgO/10 g. tissue). 

3. Evaporate to dryness on low hotplate by gradually increasing heat until dry, but 
avoid spattering. When dry, place in muffle furnace and slowly bring temperature up 
to 600 degrees C. Keep at this temperature at least 6 hours, preferably overnight. A 
fairly white colored ash should result. With organs high in blood, a yellow color due 
to iron will be present. 

4. Cool, and add with stirring 22% HCl (caution, add slowly to avoid spattering). 
Use 10 ml. for every g. of MgO used. This will give a clear solution of the proper 
acidity for Gutzelt test. (If not clear, heat to boiling on hotplate, cool and filter). 

5. Transfer to Gutzelt apparatus using 25 ml. of water. Add 2.0 ml. KI solution 
and 0.5 ml. SnCl 2 solution. Allow to stand for 20 minutes. 

6. Add 3.5 g. of Zn and place the absorption and collection tube In place; allow 
the reaction to proceed for 90 minutes, keeping the apparatus immersed In a beaker of 
tap water. 

7. A yellow to brown stain on the glass wool indicates the presence of arsenic. A 
blank control is run on all reagents used and a positive control of 5 micrograms of As. 
If the unknown is positive, a quantitative determination Is made. 

8. Prepare an oxidizing solution by mixing 8. ml. water, 1. 6 ml. of 6 N H 2 S0 4> 
and 1. 2 ml. of KM11O4 solution. 

9. 1.4 ml. of this mixture is placed in an Erlenmeyer flask (50 ml.) and the glass 
wool pledget is pushed out of the collection tube into it (using a small wooden applica- 
tor stick). A small amount of the solution is sucked up into the tube to wash any arsen- 
ic on the side of the tube into the main solution. Allow to stand 15 minutes. 






280 



10. Add 5 ml. of COLOR REAGENT and heat 15 minutes on steam bath. Cool and 
transfer to 25 ml. glass stoppered cylinder. Wash with email amounts of water. Add 
washings to solulion and finally dilute to 10 ml. mark with water. Mix thoroughly. 

11. Centrifuge and decant into a photometer euvet. Determine % transmlttance 
using 5 ml. of COLOR REAGENT diluted to 10 ml. as a blank. Read at 700 mu if 
using a Coleman Jr. Spectrophotometer. 

12. The arsenic content is determined by comparison witti a standard curve ob- 
tained by treating kn own amounts of arsenic (5, 10, 15, 20, 25, 30, 35, 40 micro- 
grams) with the above procedure. The blank is determined and, if appreciable, de- 
ducted from the amount calculated. 

Notes : 

1. Only triple distilled water (metal free) must be used for making up solutions. 

2. Ail glassware must be washed with dilute nitric acid and rinsed with triple 
distilled water (metal free) until nitric acid is removed (usually 3 rinses is sufficient) 
before use. 

Interpretation : 

1. Arsenic is not a normal constituent of the human body. However, arsenic is so 
universally found that it may frequently be present in the human body In very minute 
amounts. When the greatest quantity detected is less than 1/10 milligram per 100 gm. , 
but more than 1/50 milligram, the chemist must consult the medico-legal expert in 
charge of the case. Quantities in excess of those mentioned axe reported as signi- 
ficant. 

2. Arsenic is generally present in varying quantity in Egyptian soil. 

3. The administration of arsenic on therapeutic grounds may be a cause for 
arsenic found in the body after death. 

4. The smallest recorded fatal dose is 2 grams. Recovery has however, 
occurred after larger doses. 



281 



LIMIT TEST l'OH MERCURY ON BIOLOGICAL MATERIAL 

Reference ; 

Maren, T. H. , J. Lab. & Clin. Med. , 28, 1511 (1943) 

Princip le: 

Both a qualitative and quantitative determination for mercury Is carried out by 
forming a metal dithlzonate color complex after destruction of biological materials. 

Reagents : 

1. Sulfuric acid, concentrated, ACS 

2. Nitric acid, concentrated, ACS 

3. Hydro xylamine HC1 - 20% aqueous solution 

4. 0.25 N HC1 - 10.4 ml. analytical reagent diluted up to 500 ml. with triple dis- 
tilled water, (If this does not produce satisfactory results use 45,0430 g. of constant 
boiling HC1 diluted to 1 liter with triple distilled water). 

5. Dithlzone solution - 10 mg. per-liter - 1 ml. Is equivalent to 2.6 micrograms 
of mercury. Make by diluting 10 mg. Eastman Diphenylthiocarbazone up to 1 liter 
with chloroform, ACS. 

6. Standard mercury solution - dissolve 500 mg. metallic mercury In concen- 
trated nitric acid and dilute to 500 ml. with triple distilled water. To 10 ml. of this 
solution add 10 ml. concentrated nitric acid and dilute to 1 liter with triple distilled 
water. 1 ml. equals 0.0100 mg. of mercury (10 micrograms per ml.). 

7. Titration standard - dilute the above solution (1 ml. equals 0.0100 mg, of 
mercury) 1 to 10 with triple distilled water. 

Procedure : 

1. Weigh out 6 grams organic material into a 100 ml. long neck Kjeldahl flask. 

2. Immerse flask In cold water and slowly add 20 ml. concentrated nitric acid. 

3. After 10 minutes add 5 ml. concentrated sulfuric acid slowly; add 2 glass 
beads to prevent "bumping;" add a connecting bulb to the flask; apply heat gradually 
until full heat is used; more nitric acid must be added If charring occurs. Continue 
to heat until solution becomes clear. 

4. Add 3 five ml, portions of triple distilled water, heating to SOg fumes after 
each addition. 

Important : Cool flask before adding water e'ach time and add the water nlowlyl I 

5. Cool the clear mixture and add to the solution 75 ml. of 0. 25 N HCl and 5 ml. 
hy dro xylamine -I IC1 solution. 



282 



$. Heat to about 70° C. for 15 minutes; allow to cool Qt may be cooled under 
tlie tap. ) 

7. Transfer to 500 ml. separately funnel and dilute the solution to 250 ml. to 
bring pH to about 1. 

8. Extract with successive 2 ml. portions of dithlzone solution, shaking each 50 
times using a glass -stoppered separatory funnel. Each pure orange extract will rep- 
resent 5 micrograms of mercury. When a mixed color is obtained add standard mer- 
cury solution in 1 microgram portions (1.0 ml.) (shaking as auove) until the orange 
endpoint is reached. Calculate as below. 

a. A diluted standard control of 20 micrograms (20 ml.) 1b run through the pro- 
cedure as a positive control. 

10. A reagent blank is run with all determinations. 

Notes : 

1. All glassware must be washed with nitric acid and rinsed with triple distilled 
water until free of nitric acid (2 rinses is usually sufficient). 

2. All reagents must be made up with triple distilled water when called for. 

3. Platinum does not interfere at pH 1. 

4. 10 micrograms of mercury can be detected in the presence of 500 micrograms 
of copper. 

5. Gold gives a rose to purple color at this pH. 

6. Silver interferes both mechanically and chemically by decolorizing dithizone 
solution. 

Calculations : (Example) 

1 ml. dithizone^ 2. 5 micrograms Hg 

(ml. dlthizone used x 2.5) - ml. Hg std used = micrograms Hg 

(2 ml. x 2. 5) - 2 ml. = 3 micrograms Hg in digested sample 

Interpretation : 

1. Normal urine, blood and tissue should contain no mercury. 

2. As little as 3 grams of corrosive sublimate has been found to be fatal to a 
child and an adult. 

3. The minimum lethal dose is about 500 mg. of mercuric chloride. Organic 
mercury compounds vary widely in toxicity. 



283 



LEAD 
Blood, Tissue and Urine 






Be fere nee : 

Sandell, E.B.: Colorimetric determination of traces of metals. New York, N.Y. , 
Interscience Publishers, Inc. 2nd Ed. (1950). 

Principle : 

Lead is extracted from digestion mixtures by repeated shakings with dlthlozone 
chloroform solution. The chloroform lead dithlzonate thus formed Is extracted with 
dilute nitric acid to transfer lead to the aqueous phase. The solution Is then brought 
to a suitable pH by adding an ammoniacal citrate cyanide solution, and the lead Is 
again extracted with a portion of chloroform containing a known concentration of dithl- 
zone. The concentration of lead dlthizonate is found by spectrophotometry measurement of 
the absorption at 510 mu and referring the results to a standard curve which has been 
established under the same conditions. 

Reagents : 

1. Metal-free water: Distilled from all pyrex glass still. (Make all reagents from 
this water. Wash all glassware used in procedure with concentrated nitric acid and 
then rinse with this water. ) 

2. Concentrated nitric acid: ACS-Baker's redistilled. 

3. Concentrated sulfuric acid: Analytical reagent grade . i 

4. Chloroform: ACS-redistUled. 

5. Dithlzone: 100 mg. dlphenylthiocarbozone per liter chloroform. 

a. Solution A: Make 1:1 dilution of stock. 

b. Solution B: Make 1:10 dilution of stock. 

6. Concentrated ammonium hydroxide: Analytical reagent grade. (Low lead 
content-0.0001%). 

7. Dilute nitric acid: Dilute 10 mis. concentrated nitric acid to 1. liter. 

8. Ammonium citrate: 50 grams diluted to 100 mis. with water. Make solution 
ammoniacal (pH-9) and shake with dithlzone until lead free. Remove excess dithl- 
zone by shaking several times with chloroform. 

9. 10 per cent potassium cyanide. 

10. 20 per cent hydroxylamine hydrochloride. 

11. Ammonia-cyanide mixture: 20 grams potassium cyanide and 150 mis. concen- 
trated ammonium hydroxide diluted to 1 liter. Adjust pH to 9.5, 

12. 0. 100 per cent standard lead solution: 0. 160 grams dried lead nitrate diluted 
to 100 mis. with water acidified with nitric acid. 

14. Working lead standard: dilute 1 ml. of stock standard to 1 liter. (Equivalent 
to 1 microgram per ml.). 



284 



Procedure : 

1. Collection of (specimen: 

a. Blood: GO mis. are drawn with regular syrinrje and needle, but Immediately 
transferred to a fifty ml. centrifuge tube that has been previously washed with concen- 
trated nitric acid and metal free water. Ursc 5-10 mis. serum for each determination. 

b. Urine: 24-hour specimen is voided directly Into a 2 liter pyrex, glass stop- 
pered bottle that has been previously washed with concentrated nitric acid and metal- 
free water. Use 100 ml. urine for each determination. 

2. Digestion: 

a. Samples of the specimen Bhouldbe run in duplicate when possible. 

b. Add 10 ml. of concentrated nitric acid to each sample and digest to about 
10 ml. Add 2 ml. concentrated sulfuric acid and continue digestion until sulfur trl- 
oxide fumes are given off. (If charring occurs add more nitric acid as needed to clear). 
Add 3 ml. water to each sample three times, digesting to fumes between each addition. 

c. A standard (10 ml. working standard) and a reagent blank (10 ml. metal -free 
water) are taken through digestion process. Exact quantities of all reagents are added 
to all digestion flasks. 

3 . Isolation of lead: 

a. Rinse digest into a glass -stoppered separatory funnel with three 5 ml. por- 
tions of ammonium citrate. 

b. Add 1 ml. hydroxy lamlne hydrochloride and five drops thymol blue indicator. 

c. Add concentrated ammonium hydroxide until solution is basic to litmus. Add 

5 ml. potassium cyanide and adjust pH to blue color of Indicator with ammonium hydrox- 
ide. 

d. Add 2-5 ml. dithizone solution A and shake for 15 seconds. Draw off dithi- 
zone into a separatory funnel and continue to extract until there Is no mixed color 
formed. 

e. Shake the combined extracts with 10 to 20 ml. water containing 1 drop of 
50% ammonium hydroxide. Draw off dithizone and extract the ammonia solution with 1 
to 2 ml. of dithizone A and add this to the main extract. 

f . Shake the dithizone extract with 15 ml. dilute nitric acid for 30 seconds and 
transfer the aqueous phase to a separatory funnel. Extract again with 10 ml. dilute 
nitric acid and combine the aqueous portions. Shake several times with chloroform to 
remove excess dithizone. 

g. Add 6 ml. ammonia-cyanide mixture and 20 ml. dithizone solution B. Shake 
for 1 minute. Read a portion of the dithizone layer with a spectrophotometer at a wave- 
length of 510 millimicrons using 10 x 75 mm. square cuvets, and using dithizone solu- 
tion B as the blank. 

Calculation s: 

1. Per cent transmlttance of the 2 unknowns , reagent blank, and the standard are 
compared with and read In micrograms of lead from standard curve. Report as mg. 
per cent lead. 



285 



Example: 

a. Per cent trans' mi. ttance unknown number 1 - 85 « 2.5 micrograms 

b. Per cent transmittance unknown number 2 = 94. 5 = 3 micrograms 

c. Per cent transmittance reagent blank a 89 s 1 microgram 

d. Per cent trans ml ttance standard * 78 s 11 micrograms 

f . Knowing that the reagent blank read 1 microgram and that the standard 
originally contained 10 micrograms, subtract 1 microgram from the average of the 2 
unknowns, = 1. 75 micrograms. This is the amount of lead In the sample taken. 

Standardization of Curve : 

1. A series of dilutions from 5-10-15 through 50 micrograms are prepared 
from the stock standard. These are taken through the entire procedure and the results 
are plotted. 

Notes : 

1. All reagents used In procedure must be prepared from metal-free water. 

2. All glassware used In procedure must be washed with concentrated nitric acid 
and rinsed thoroughly with metal-free water. Traces of nitric acid remaining In glass- 
ware will destroy the lead dlthlzonate color complex. 

Interpretation : 

1. Normals: 

a. Urine: 2.5 to 3.5 micrograms per 100 mis. 

b. Whole Blood: 0.001 to 0.006 mg. per 10 ml. sample. 

(1) Blood cells: 0.002 to 0.11 mg. per 10 gram sample, 

(2) Blood serum: 0.000 mg. per 10 ml. sample. 

2. Acute lead poisoning: 

a. Urine: up to about 300 micrograms daily. 

b. Whole blood: in lead poisoning symptoms begin at 30 micrograms per 
100 ml. with severe symptoms above 60-100 micrograms per 100 ml. 






286 



BARBITURATES 
Blood, Urine, Tissue 

Reference: Goldbaum, Leo R. , Anal. Chem. 24, 1604-1607 (1952) 

P rinciple : 

This procedure is a simple, rapid, ultraviolet spectrophotometric procedure for 
the specific Identification and quantitative determination of micro quantities of bar- 
biturates, ft Is based on the observation that barbiturates have characteristic absorp- 
tion bands in strong alkali and different characteristic absorption bands at a pH of 
approximately 10.5. A barbiturate is Indicated only when a maximum positive differ- 
ence appears at 260 mu. which decreases to a negative difference at about 250 mu. 
A quantitative estimation can be made from the difference at 260 mu. The variations 
in the optical density differences at 260 mu. due to normal absorbing substances will 
produce less than a 5% error. As the barbiturate concentration decreases the error 
will increase. As little as 1. microgram of barbiturate can be detected per milli- 
liter of blood and 3. 3 micrograms par gram of tissue. 

Apparatu s: 

Beckman DU quartz photoelectric spectrophotometer, one centimeter quart* 
cuvettes, Waring blendor, pH meter. 

Reagents : 

1. Borate Buffer . Dissolve 12.369 grams of boric acid and 14.911 grams of 
potassium chloride in water and dilute to 200 milliliters to prepare a 1 molar solution 
of these salts. Filter off any undissolved salts after standing at room temperature 
for 24 hours. 

2. Sodium hydroxide solutions . 

a. Prepare an approximately 0.45 normal sodium hydroxide solution. The 
normality Is adjusted with a pH meter until a pH of 10,5 Is obtained when two parts 
of alkali are added to one part borate buffer, 

b. Prepare an approximately 0.90 normal sodium hydroxide solution from 
saturated sodium hydroxide. The normality is adjusted with a pH meter until a pH 
of 10.5 is obtained when two parts of alkali are added to two parts borate buffer. 

3. Solvent s. Wash reagent grade chloroform successively with 1 normal sodi- 
um hydroxide and twice with water. For each liter of solvent, use 100 milliliters 



28T 



of wnsh solution. Wash only the volume required for dally use. On standing chloro- 
form tends to decompose, 

4. St andard soluti on. Prepare a standard solution of phenobarbltal which con- 
tains 20 micrograms per milliliter, 

5 - Phosphate buffer. Prepare a 0. 1 molar phosphate buffer with a pH of 7.4. 
Mix 39. 50 ml. 0. 1 N NaOH with 50 ml. 0. 1 M ICH 2 P0 4 and dilute to 100 ml. in a 
volumetric flask. 

Procedure: 

A. BLOOD or PLASMA 

1. Oxalated blood or plasma is adjusted to pH 4 to 6 with 0. 1 N HC1 and ex- 
tracted with chloroform in a separatory funnel. Use 50 ml. chloroform for samples 
of 1 to 5 ml. Use 75 ml. chloroform for samples of 5 to 10 ml. 

2. Prepare a standard control using the same volume of specimen plus one ml. 
of the phenobarbltal standard solution. 

3. Filter the chloroform extract through No. 41 Whatman filter paper. A clear 
aliquot of 40 to 60 ml. is obtained. 

4. Extract the filtrate with 4 ml, 0,45 N NaOH using a dry separatory funnel. 

5. Discard the chloroform layer and collect the aiicali layer In a small tube. 

6. Centrifuge the alkali extract and transfer 3 ml. of clear solution to a 1 cm. 
quartz cuvette. 

7. Take optical density readings at 230. 240, 250, 260, 270, 280, and 305 mu 
against a blank of the alkali. 

8. Take 2 ml. of the alkaline extract from the cuvet and mix with 1 ml. of borate 
buffer. The final pH should be between 10. 2 and 10. 6. 

9. Transfer the buffered extract to a cuvette and take optical density readings 
at the v/ave lengths indicated In (7) above. The blank Is prepared by mixing 2 ml. 
of alkali and 1 ml. of borate buffer. 

1C. These last optical densities are corrected for dilution with the buffer by 
multiplying by 1.5 and then subtracted from the optical density of the alkaline extract. 



288 



B. URINE 

1. Take 1 to 5 ml. of urine with the pH adjusted to between 4 and 5 with 0. 1 H 
HCl and extract with 50 ml. of chloroform. 

2. To remove interfering substances, shake the chloroform extract with 5 ml. 
1 M phosphate buffer of pH 7. 4 using a clean separator/ funnel. 

3. Filter the chloroform layer through a Whatman #41 filter paper. 

4. Take the filtrate and proceed as under BLOOD. 

C. TISSUE 

Tissues are prepared for extraction by homogenizing with a mi n im u m amount of 
distilled water in an all -glass Waring type blendor. Chloroform is the solvent of 
choice for all tissues except brain, where ether or ethylene dichloride can be used. 
A weighed sample of tissue may be homogenized and made up to a specified volume, 
using an aliquot for analyses; or a small sample may be homogenized and quantita- 
tively transferred Into a separate ry funnel containing the solvent. For the extraction 
of 5 ml. of homogenate, containing 1 or 2 gram sample, 75 ml. of solvent are used. 
The analysis is continued as described for blood except that the aliquot of clear fil- 
tered solvent is extracted with approximately O.DK sodium hydroxide. The normal- 
ity of the alkali Is increased to eliminate the turbidity that sometimes occurs with 
weaker alkali. The adjustment of pH is made by adding an equal volume of boric 
acid-potassium chloride buffer to yield a pH between 10.5 and 10. 2. The optical den- 
sities of the buffer solution are multiplied bv 2. to correct for dilution with the 
buffer, then nubtracted from those of the strong alkali. 

Calculations : 

Micrograms of barbiturate per ml. or gm. = ^ * . 

a. Micrograms of barbiturate added to known standard sample. 

b. Optical density difference of unknown extract in alkali and In pH 10. S 

at 260 mu, 

c. Amount of sample. 

d. Optical density difference of standard extract In alkali and pH it. t at 
260 mu. minus (b) above. 



289 



EXAMPLE: 









Optical Density Readings 




























Standard Control 






Unknown 




Wave 




PH10.S 




Differ- 








Differ 


length 


Alkaline 


buffer 


xl. 5 


ence 


Alkaline 


10.6 


x 1.5 


ence 


230 


.670 


.392 


.588 


.082 


.800 


.498 


.748 


-.062 


240 


.470 


.428 


,552 


-.082 


.600 


.490 


.735 


-.135 


2S0 


.470 


.307 


.465 


.005 


.540 


.361 


.542 


-.002 


260 


.465 


.164 


.276 


.189 


.495 


.259 


.388 


.107 


270 


.380 


.170 


.255 


.125 


.412 


.247 


.370 


.042 


280 


.305 


.171 


.257 


.048 


.350 


.243 


.366 


-.015 


290 


.250 


.153 


.230 


.020 


.293 


.210 


.315 


-.022 


305 


.130 


.082 


.123 


.007 


.160 


.110 


.165 


-.005 


a 


20 micrograms 














b 


.107 






•20 ug 
6 ml. 


x.107 
x .082 


a 5.22 


ug/ml. 




e 


5 ml. blood 
















d 


.183 - .107 


= .082 














NOTES: 



















( 



1. The optical density at 305 mu is an indication of the amount of absorbing 
substances other than barbiturates. The differences at this wave- length should be 
insignificant. 

2. The sample used for analysis should be such that the alkaline extract should 
contain about 25 micrograms of barbiturate per ml. 

a. Salicylates do not interfere. 

4. Dlcoumarol, dilantln, and sulfadiazine appear In the alkaline extract but do 
not Interfere because they have the same absorption In alkali and in pH 10. 5 solution. 

5. The sodium hydroxide solutions can be stored in polyethylene bottles without 
decomposition. 



290 



Interpretation : 

After a therapeutic dose of a barbiturate the blood level may be 1 to 10 micro- 
grams per ml. The corresponding tissue level Is higher. The toxic level ranges 
from 15 to 100 micrograms per ml. The effective dose of barbiturates varies 
greatly with the particular compound used, as does also the effective concentration 
In tissues. Since the toxic level of one barbiturate may be a safe level for another, 
it is important to be able to Identify the particular barbiturate in question. A paper 
chromatographic method of Identification is given in this manual p. 292. 



291 



PAPER CHROMATOGRAPHY FOR THE mENTIFICATION OF 
THE COMMON BARBITURATES IN BLOOD, URINE, AND TISSUE 



Reference : 

Algeri, E. J., and Walker, J. T.; Am. J. Clin. Path., 22, 37-40 {1952). 

Principle: 

A paper chromatographic method la used for the specific identification ot the 
common barbiturates. This technlc Is used In con Junction with the spectrophotometry 
procedure previously outlined. For a given solvent and temperature, a substance has 
a characteristic Rj value; its determination serves as a means of Identification. 

Apparatus : 

1. Museum Jars approximately 18 inches high and 8 inches wide equipped with 
rubber gasket, lid, and screw clamp. Stopcock grease may be used as an air seal. 

2. Ultra-violet lamp. 

3. Whatman No. 1 filter paper 17 X 14 inches. 

Reagents : 

1. Solvent - n- Butyl alcohol Is saturated with 5 Normal ammonium hydroxide 
by shaking m a separator/ funnel and allowing the two phases to separate. The 
aqueous portion Is then drawn off Into a small beaker and* placed la the bottom of 
the tank, separate from the surrounding saturated mobile organic phase. This 
solution should be freshly prepared. 

2. Color developer 

a. Silver acetate - A 0. 1 per cent aqueous silver acetate solution Is prepared. 

b. s-Dlphenyl carbazone - A 0.1 per cent s-dlphenyl carbazone solution In 
absolute alcohol. 

3. Standards - Standard barbiturate solutions are made up in acidified water 
(1:500 H 2 S0 4 > to contain 1.0 milligrams per milliliter. 

Procedure : 

1. The combined alkaline extracts containing a barbiturate (from the quantita- 
tive procedure, p. 286) is acidified with dilute hydrochloric acid and extracted at 
least twice with ether. 

2. The ether extract is reduced in volume by evaporation. 

3. Approximately 0.01 ml. of the extract is applied to the filter paper cylinder 
at a base line 3 cm. from the bottom. 



202 



A. A|»i»ly 0.01 tnl. (10 mlcrogramit) of the known barbiturate HlandardH at 3 cm. 
lull- rva In lo Dm; j»a|H'r. 

li. Hull tlit; paper cylindrical ly and nlaplc no Dial the edged do not touch, 
0, I tint-', ""■ l>"|M?r cylinder In the lank containing tl»c Hoivcnt for one hour, 

7. l<owcr I he p:i|ier cylinder Into the nolvcnt. 

8. Place the nytilum In a dark cabinet ill conntant. temperature for 4 hours. The 
tin) vent front nhould move at leant 120 mm, 

0. Dry the chrdmalogram rapidly and uniformly In air to minimize the spreading 
of lite Hpotn. The Htnplcri are removed. 

10. locate the Hfiotii by placing a pencil dot In the center of the dense area, while 
ohnervlng under ultra-violet light. 

11. The wheel Ih dipped In 1 per cent silver acetate bo lull on for one -half minute, 
waiihed four inlnuleH In running water, and Immersed directly In 0.1 per cent s-dlph- 
enyl carba/.one In OH per cent ethyl alcohol for one minute. The spots appear deep 

In own In color against a light pink background. 

12. Determine the It* valucH for each spot. 

CalculatlotiH: 



|i _ Distance moyed_by solute 
Distance moved by solvent 



Kxample: 





Dlntance 


Itarlillurale 


of Holute_ 


Seconal 


112 mm. 


barbital 


78 


tin known 


83 


I'lienouarbltal 


83 


Amytal 


100 


Nembutal 


100 



Dlntance lt, 

of Holvenl 



132 mm. .880 

131 .695 

131 . 034 

130 .038 

131 .832 
131. G .750 

The It value of Die unknown after four bourn running time coincides with that of 
phenol) arlmal. 

Notes: 

I. l>eiilolhal in revealed under ultra-violet light without being developed. 



293 



SIMPLE BLOOD TESTS AVAILABLE TO THE GENERAL PRACTITIONER 



Reference : 

Mandel, E. E.. M.D. , Lehmann, E. B. , M.D, , Paris, D. A., M.D., U.S. 
Public Health Service. Communicable Disease Center, Federal Security Agency. 
March 1950. 



Blood Tests: 









Sedimentation Rate 
Icterus Index 

"H" Flocculatlon 

Creatinine 

Glucose 






Reagents ; 



Potassium ammonium oxalate solution: 

Ammonium oxalate 1. 2 g. , potassium oxalate 0. 8 g. . distilled water, dilute up 

to 100.00 cc. Use 2 drops (dried) for each ml. of blood. 
Saturated solution of picric acid prepared at 2*° C. 
10% sodium hydroxide. 

Distilled water. ( 

Hayem's solution - Mercury bichloride 0. 25 gm. , sodium sulfate Z. 60 gm. , sodium 

chloride 1.00 gm. , distilled water, dilute up to 100.0 cc. 



Appa ratus: 

—EC 

12 pyrex test tubes, without Up, 100 x 13 mm. , graduated at 1, 2, 4, and 5 cc. , 

and several dozen identical test tubes, nongraduated. 
Test tubes of identical size containing standard solutions of potassium dichromate, 

all stoppered and sealed with paraffin. 
Small funnels and filter paper (Whatman No. 1). 
Medicine droppers. 

Beaker or small pot, stand and bunsen burner or electric hot plate for water bath. 
3-slot comparator with ground-glass background, approximately 3x2 1/2 x 1 inch 

In dimension. 
Desk lamp with daylight bulb 
Set of Westergren sedimentation tubes. 

Princi ple; 

A knowledge of the physiological basis for the Importance of these tests will be as- 
sumed, and only the procedures will be described. Routine analysis of the blood and 

294 



urine is an invaluable adjunct to (he clinical examination, Yet the equipment, time and 
technical skill required for many standard blood tents make their performance In the 
average practitioner's office impractical, m addition, a clinical laboratory may be too 
distant or Its fees too high for the patient to afford. Therefore, it has seemed highly 
desirable to find methods that arc "so simple and mechanical in their operation that 
the possibility of large error is excluded and so direct that the results are presented 
as pictures whose significance can be read at a glance." If such tests could be per- 
formed by the doctor or his nurse with little loss of time, the results would constitute 
a valuable supplement to the physical examination, helpful both In the ready detection 
of dlsenBe and in the follow-up of patients. 

A. Collection of Blood Samples : The five simple blood tests to be described, used 
either singly or as a "battery, " or in combination with cell count and other procedures, 
will provide quick and valuable diagnostic information with a minimum of equipment, 
expense and technical experience. They can be all carried out on a single sample of 
venous blood. Only about 7 cc. Is required, this being- divided into two parts. Five cc. 
Is allowed to coagulate In one tube to provide serum for Ilayem's test and Icterus Index, 
and 2 cc. is mixed in another tube with the dried residue of four drops of oxalate solu- 
tion for the sedimentation rate, creatinine and glucose determination, and also for 
blood count if desired. 

B. Sed iment atio n Rate: Using the Westergren tube as a pipette, oxalated blood Is 
sucked up to the zero mark. At the end of one hour the height of the boundary between 
the packed cells below and the supernatant plasma above is read from the calibrations 
on the tube. A drop of the level of the cell column of more than 10 mm, in men or more 
than 15 mm. in women in one hour is suggestive of disease. 

Erroneous results may be obtained if there is prolonged venous stasis on with- 
drawing blood, traces of water, alcohol, ether and other substances In syringe, needle 
or tube, or delay of over six hours In conducting the determination. Results will also 
be affected by exposing the specimen to temperatures considerably above or below aver- 
age room temperature, and by racking the tube In a position that is not exactly perpen- 
dicular. 

C. Creatinine: The urine and blood levels of creatinine are essentially Independ- 
ent of the amount of protein consumed and the time of food Intake. This is in contrast 
to urea, the blood concentration and urinary elimination of which are greatly influenced 
by these physiologic factors. 

To 4 cc. of a saturated solution of picric acid add 1 cc. of oxalated blood, using 
syringe or medicine dropper. Shake and let stand 5 minutes. Filter Into an identical 
test tube until the clear yellow filtrate reaches the 1 cc. mark. Add 1 drop NaOH. Shake. 
In 15 minutes compare with standard solutions. An Increase of 3 mg. per cent or more 
can be detected readily without the aid of a comparator. 

D. Glucose; Same solution that has been used for the creatinine determination Is 
immersed in boiling water for about 3 minutes. Distilled water is added to the 5 cc. 
mark. Shake and compare with the standard solutions. If color Is more Intense than 
2400 mg. per cent dtchromnte standard, tranBfer 1 cc. lo another tube and dilute with 

295 



1 cc. distilled water. After dilution the value obtained by color comparison must be 
doubled to obtain actual glucose level. Further dilution to the 4 cc. mark Is necessary 
If blood sugar le above 400 mg. per cent. 

A coincidental rise In creatinine Increases the intensity of the plcrate glucose 
color reaction sufficiently to warrant Its consideration in blood specimens containing up 
to 200 mg. per cent of glucose. For every milligram per cent of creatinine found in ex- 
cess of the average normal of 1 mg. per cent, 2 mg. per cent of glucose should be de- 
ducted from the final glucose value. This may well be Ignored, however, for the sake 
of convenience and In such undertakings as screening for diabetes since the coincidence 
of azotemia and diabetes Is Infrequent and the error would be on the plus side: it would 
tend to add an occasional uremic individual to the hyperglycemic group but would not 
cause any diabetics to be missed. 

The visual tests for creatinine and glucose have been round useral as ready 
checks on doubtful results of the precision methods. They are suitable for front-line 
practice In the absence of routine laboratory facilities and they afford Information that 
Is adequate for many clinical purposes. 

E, Icterus Index: The icterus Index may be determined by direct comparison of 
the color of either plasma (oxalated blood) or serum (coagulated blood) with the tubes 
containing standard solutions of potassium dlchromate. Intensity of the yellow color of 
the sample In excess of a 70 mg. per cent dlchromate solution Is an abnormal finding. 
The test Is of value In detecting abnormal amounts of blood bilirubin even In the absence 
of clinically apparent jaundice, without differentiating between the various types of 
Jaundice . 

F. Hayem's "H" Test : The finding of serum protein alterations is indicative of 
organic disease. The various flocculation tests In general use constitute, in essence, 
practical qualitative substitutes for the exact measurement of the various serum pro- 
tein components by .chemical, electrophoretlc and other means. While used largely as 
"liver function tests", they may be employed advantageously In other diseases, espe- 
cially those of infectious or neoplastic origin. Positive reactions usually are taken to 
reflect hypoalbuminemia and/or an Increase In one or more globulins. 

The flocculation test with Hayem's solution Is one of the simplest and quickest 
In this category. As a nonspecific screening test it was found to be more sensitive than 
the Takata-Ara, cephalln-cholesterol flocculation, and formol-gel reactions, and about 
equal to the thymol turbidity test; it was not positive as often as the sedimentation rate 
and the zinc sulfate turbidity method. In testing for primary hepatic disease, the "H" 
reaction was surpassed in sensitivity only by the sedimentation rate, thymol and zinc 
sulfate turbidity tests. It was positive in all examined cases of severe or prolonged 
hepatitis of hepatic cirrhosis, and of active rheumatoid or Infectious arthritis; further- 
more it was positive In 61 per cent of ftcttte-infections, 47 per cent of chronic lung dis- 
orders and 59 per cent of cancer. 

A positive "H" flocculation Implies systemic disease (metabolic, Infectious or 
neoplastic) usually of a more serious and/or advanced nature than that Indicated by an 
Increased sedimentation rate. It may be the first abnormal finding in such conditions as 
cirrhosis, syphilis, pyonephrosis, tuberculosis or metastatic neoplasm. The test has 
been found helpful in such problems of differential diagnosis as obstructive vs. hepatic 

296 



Jaundice, tumor 01 the right kidney vb. hepatomegaly, epIutaxlH due to local lesion vb. 
chronic hepatic dlaease, chtlecyHtopallty vu. neopia/jtlc liver Involvement, "rheuma- 
tism" va. rheuma'.nJd arthritis, congentlvc vs. Infectious pleural effualon, nutritional 
vs. leukemic anemia. In each of these caa-jB a positive reaction favors a hepatic, in- 
fectious or neoplastic cause of the condition. 

The Hayem teat may serve as a prognostic adjunct to the sedimentation rate 
and as a practical substitute for more elaborate tests In the screening for, and man- 
agement of, diseases affecting the retlculo -endothelial syiitem. 

Procedure : Equal volumes of serum (from coagilated tube) and Hayem' b 
solution - usually 1 cc. of each - are mixed. Development of a precipitate within 1 to 
24 hours Is considered a ponltlve reaction (graded as 1, 2 or 3/). An Immediate pre- 
diction of the eventual outcome can usually be made by overlaying the serum with 1 
drop of Hayem '8 solution. A faint transient clouding may occur In normal serum, but 
a well discernible "reversible flocculation" Is abnormal. 



BLOOD VALUES CORRESPONDING TO EACH OF THE STANDARD SOLUTIONS 



Potassium 
Dlchromate 

% 


.05 


.07 


.10 


.16 


.25 


.30 


.50 


.70 


.90 


1.0 


1.2 


1.5 


1.9 


2.4 


3.0 


Glucose 

mg.% 








50 


78 


90 


112 


130 


145 


150 


160 


175 


185 


200 


215 


Creatinine 
mg.% 








0.8 


1.8 


2.0 


3.2 


4.2 


5.0 


6.4 


6.0 


7.1 


8.0 


9.8 


14 


Icterus 
Index 
units 


5 


7 


10 


IS 


25 


30 


50 


70 


90 


100 


120 


150 


190 


240 


300 



297 



ROUT INK C1IKMICAL ANALYSIS OV URINARY CALCULI 



( 



Reference : 

Winer, J. II. , Malllce, M. R.: Routine Analysis of Urinary Calculi. A simple 
rapid method using spot tests. .F. Lab. & Clin. Med. 28, 898-904 (1942). 

Prin ciple; 

After preparing a physical description of the calculus as to weight, size, shape, 
color, surface appearance, and consistency, the calculus is finely pulverized in a 
mortar for chemical analysis. This analysis is performed using a series of black 
and white spot plates, and the characteristic groups tested for by means of simple 
spot tests. 



Appa ratus : 

1 12-hole white spot plate 

2 3-hole black spot plate 

Reagents; 

1. Sodium Carbonate: Dissolve 20 grams of the anhydrous salt in 80 ml. water. 

2. Uric A cid Reagent: Dissolve 10 grams pure sodium tungstate (NagWC^) 

in about 60 ml. water; add 5 grams pure arsenic pentoxide, 2.5 ml. of 85% 
phosphoric acid, and 2 ml. concentrated HC1. Boil mixture 20 minutes, cool, 
and dilute to 100 cc. with water. Filter if solution is cloudy. 

3. Ammonium Molybdate : Dissolve 3.5 grams of the salt {NH^gMOyC^- 4H 2 In 
74 ml. water, add 25 ml. concentrated nitric acid, and shake until dissolved, 

4. Am m oniurn Jjydrgxidej Concentrated, Reagent grade. 

5- Sodium Cyanld°: Dissolve 5 grams in 100 ml. water, and add 0.2 ml. ammo- 
nium hydroxide as a preservative. Store solution in refrigerator. 

6. Sodiu m Nltro prusslde : Dissolve 5 grams in 100 ml. of water. Discard when 
color fades. 

7. Nitric Ac id: Concentrated, Reagent grade. 

8. Ammonium Sulfocyanate : Dissolve 3 grams of the salt (NH^SCN) in 100 ml. water. 

9. Hydrochloric Acid: Add 10 ml. of concentrated HCl to 90 ml. of water. 

10. Sodium Oxalate: Saturate 100 ml. of water with the Bait by adding 5 grams a 
and allowing to settle. 

11. Sodi um H ydroxide: Dissolve 20 grams of the Reagent grade pellets in 80 ml. 
of water. 

12. Titan Yello w Dye: (also called "Clayton Yellow"). Dissolve 0. 1 grams of dye 
in 100 ml. water. Make the solution alkaline with 3 drops of 20% sodium hy- 
droxide (see No. 11). Stor^ in am amber bottle. Prepare fresh every 30 days. 



298 






13. Ne aster's Rea gent : Dissolve 22.5 grams of iodine in 20 ml. of water contain- 
ing 30 grams of patasnlum iodide. Add 30 grams of pure metallic mercury. 
Shake tte mixture well, cooling frequently under tap water until yellow color 
due to iodine disappears. Decant the supernatant aqueous solution. Test a 
portion of it by adding a few drops of the reagent to 1 ml. of 1% soluble starch 
solution. Test Bhould be positive (a purple to black color). Dilute with water 
to about 200 ml., mix well, and add to 975 ml. of 10% sodium hydroxide. Mix 
well and allow to clear by standing. 

14. Manganes e Dioxide : powdered, Reagent grade. 

15. Barium Chloride: Dissolve 5 grams of the salt in 100 ml. of water. 

16. Sodium_Ni trite: A fresh solution, 0.1 grams of the salt per 100 ml. of water, 
should be prepared weekly. 

17. R eagent " S"; Dilute 1 ml. of dimethyl -a-naphthylamtne (Eastman) In 250 ml. 
of 95% alcohol. 

18. Chl orofor m: pure, Reagent grade. 

19. Acetic A nhydrid e: concentrated, Reagent grade. 

20. Sulfuric A cid: concentrated, Reagent grade. 

Procedure : 

1. Weigh the stone on the analytical balance to the nearest one-tenth milligram. 
If stone has been freshly taken or has been in preservative, allow to dry at 
room temperature for at least 24 hours before beginning this procedure. 

2. Next, examine the stone in a good light. With iha aid of a hand lens note ths 
color, shape, and surface appearance, (See notes 1-2). 

3. Using a metric ruler, measure the stone's length, width, and thickness to 
the nearest millimeter. 

4. ha small clean mortar pulverize about 60 mg. of the stone, noting its 
consistency, (See note 3). 

5. Refer to Table 1 and first complete this routine spot test battery. Perform 
all spot tests on a white spot plate unless otherwise indicated In the table* 

(Starred (*) tests are to be performed on black spot plate ). Note all 
positive reactions, 

6. If no positives have been found in Table 1, or if one of the rarer calculi Is 
suspected, next refer to the "Table of Special Tests," Table 2 for a more 
complete analysis. 



Z99 



TABLE NO. 1 
Chemical Group 



ROUTINE TESTS ON URINARY CALCULI 
Kcngeuta Added HonultB 



( 



1. URATES 



2. PHOSPHATE 



3. IRON 



4. CYSTINE 



Pulverized atone 

1 drop Na 2 C0 3 

2 drops uric acid Reag. 

Pulverized atone 
6 dropa ammonium 
molybdate 

Palverized atone 

3 drops nitric acid 
3 drops ammonium 

thiocyanate 

Pulverized stone 
1 drop NH 4 OH 

1 drop sodium cyanide 
Walt 5 minutes 

2 drops sodium nftro- 
prusslde 



Prompt deep blue color 
(pale blue io negative) 

Bright yellow precipi- 
tate 1b positive 
(allow 5 minuter) 

Red color is positive 
for iron 



Beet-red color Is 
positive for cystine. 
(May fade to red-orange) 



Dissolve 25 mg. of the remaining pulverized atone in 2 ml, of 10% hydrochloric 
acid fa a small test tube. Stir for 2 minutes. 



5. CARBONATE 

6. CALCIUM* 

7. MAGNESIUM 

8. AMMONIA 

9. SULFATE* 
10. OXALATE 



Observe the HCl solution 
of calculus for bubbles. 

3 drops acid extract 

4 drops sodium oxalate 
3 drops NH^OH 

3 drops acid extract 

3 drops sodium hydroxide 

1 drop Titan Yellow Dye 

3 drops acid extract 

3 drops sodium hydroxide 

4 drops Nessler'a Reagent 

3 drops acid extract 

4 drops barium chloride 

To the remaining acid ex- 
tract in ths teat tuba add 
a pinch of manganese di- 
oxide. 



Effervescence is posi- 
tive for carbonate. 

White precipitate is 
positive for calcium. 

Blood red precipitate la 
positive (any other color 
1b negative) 

Yellow-orange precipi- 
tate la positive for 
ammonium 

White precipitate 1b posi- 
tive for sulfate 

Tiny bubbles of gas "ex- 
plosively released" la 
positive for oxalate. 



( 



300 



TABLE NO. 2 - SPECIAI TESTS FOR HAHlilt URINAR\ CALCULI 
Chemical Group Reagents Added Results 



1. XANTHINE 



Pulverized stone 
Dissolve in nitric acid 
Dry In evaporating dish 

on water bath 
Add 3 drops ammonium 

hydroxide 

Pulverized stone 
5 drops chloroform 

Pulverized stone 

5 drops chloroform 

10 drops acetic anhydride 

1 drops sulfuric acid 

Pulverized stone 

2 drops HC1 

2 drops sodium nitrite 

3 drops ammonium sulfa- 

mate 
3 drops Reagent "S" 



7, After completing the spot test analysis, report all of the physical and chemi- 
cal findings In the following manner. 



2. INDIGO 

3. CHOLESTEROL 

4. SULFONAMIDE 



Yellow residue which 
turns orange when the 
ammonia is added is a 
positive test 



Blue color is positive 
for indigo 

Play of color ending In 
green Is positive for 
cholesterol 



Red color is positive 
for sulfonamides 



NAME John Doe 



WARD 126 



DATE 12/10/57 



Source of Calculi: Renal 



Weight 


67 . 5 mg. 


Size 


5x4x3 mm. 


Shape 


round 


Color 


red-brown 


Surface Appearance roughened 


Consistency 


hard, brittle 


Chemical Analysis 
Category of Stone 


Ca, M K) C0 3 , C ? 4 
OXALATE -CARBONATE TYPE 



301 



NOTES: 






( 



1. The shape, weight, and size of a particular calculus la quite Independent of 
the type of atone. In reporting shape the calculus is iIcHcrJbcd ob being cither 
oval, round, flat, cubic, pyramidal, or Irregular. 

2. The color, surface appearance, and consistency of the calculus are closely 
related to the type of stone. LlBted In Table 3 arc the most common charac- 
teristics of various types of calculus. A study of the physical characteristics 
Is often helpful in confirming a doubtful positive chemical test. 





TABLE 


NO. 3 - PHYSICAL C 


HARACTERISTICS OF 
Surface Appearance 


CALCULI 


Type of Calculus 


Color 


Consistency 


Urate 




yellow to dark red- 


slightly rough 


quite hard 






brown 






Phosphate 




white, gray, to 


smooth, sandy 


chalky, brittle 


_ 




yellow-gray 






Cystine 




pale yellow 


waxlike 


soft 


Carbonate 




dull white to red- 
brown 


chalky 


usually quite hard 


Oxalate 




white, grey, or 
red-brown to 
black 


tube re ul ate d or 
quite rough 


very hard 


Xanthine 




brown 


smooth, wax-like 


soft 



( 



In about 40% of the stones submitted for analysis, the quantity Is not suffi- 
cient to provide 60 mg. of material for analysis. Therefore it Is best to 
test for the presence In these cases' of calcium, phosphate, and oxalate first, 
before proceeding with the other tests. Stones weighing less than 5 mg. are 
almost impossible to analyze correctly by this method. 



302 






TABLE NO. 4 - FREQUENCY OF OCCURENCE OF THE VARIOUS 
CHEMICAL CONSTITUENTS IN CALCULI* 
Chemical Group % Occurrence 



Calcium 

Phosphate 

Oxalate 

Urate 

Carbonate 

Magnesium 

Ammonium 

Cystine 

Iron 

Sulfate 

Xanthine 

Cholesterol 

Indigo 

Sulfonamides 



97% 

88% 

65% 

15% 

12% 

25% 

20% 

2% 

Rare 

Rare 

0.5% 

Rare 

Rare 

Rare 






♦Based upon a study of calculi analyzed by this laboratory through the 
year of 1956. 



. 



303 



CIIOICK OF LAUOUATOHY TESTS 



Reference : Pcnin II. Long, M.D. , Modern Medicine, November 1, 1354. 

ExcckhIvc rbllance on laboratory teats la expensive for the patient and, by over- 
loading the tccliniclan with work, Impairs accuracy In the laboratory. Physical exam- 
ination and determination of previous disease of the patient often obviates the need for 
many laboratory procedures. 

Hemoglobin, leukocyte count, and differential count from a stained smear should be 
determined at the initial examination of a patient. The sedimentation rate Is measured, 
and the feces are tested for occult blood with gualac. Sickle-cell preparations are made 
for new Negro patients. Urinalysis should Include specific gravity, albumin, sugar, and 
acetone measurements and microscopic examination of the sediment. 

Serologic reactions for syphilis are often done at the first examination. Spinal fluid 
of patients with syphilis is studied at least once. 

Blood urea nitrogen or nonprotein nitrogen is measured in a patient with albumin- 
uria, progressive hypertension, changes in the retinal arterioles, or an enlarged pros- 
tate. The fasting blood sugar is determined for patients with glycosuria or suspected 
hypoglycemia and as an aid in the management of diabetes. 



( 






Knowledge of serum electrolyte concentrations is valuable with dehydration, vomit- 
ing, and diarrhea. The carbon-dioxide combining power of the blood and serum levels 
of chlorides are helpful in treatment of diabetic acidosis, pulmonary insufficiency, and 
severe renal disease. Sodium concentration is measured when Addison's disease is 
suspected and during therapy by salt restriction and diuretics. Potassium levels should 
be determined when parenteral fluids are Injected or when gastric suction is continuous, 
and for certain types of renal Insufficiency. 

In the differential diagnosis of jaundice, the serum bilirubin is more valuable than 
the icterus index, and the alkaline phosphatase level provides additional help. The thy- 
mol turbidity is more useful than the cephalln flocculatlon in assessing hepatocellular 
damage. Bromsulphalein retention is accurate when the patient is not jaundiced. 

Total proteins are measured in the study of malnutrition, anemia, ascites, and 
edema. Determination of the albumin and globulin should be deferred until the total 
proteins are known, unless multiple myeloma is suspected. 

Use of laboratory procedures should be limited to those specifically indicated. 



304 



CLINICAL INTERPRETATION 



Determination 



Normal Range 



Amylase (serum) 50-200 units/100 ml. 



Basal Metabolic Rate -10 to /15% of 

average normal 



PlBcueaion of variations In Disease States 

Increased values are seen In diseases in- 
volving organs secreting amylase. For ex- 
ample; acute and chronic pancreatitis; 
secondary pancreatitis; and mumps. 

BMR is elevated in: 

1, High calorie diet; 2, Fever; 3. Hyper- 
thyroidism; 4. Other endocrine disorders. 
The BMR la decreased in the opposite con- 
ditions. In addition, apprehension or 
nervousness can elevate the results 20- 
30% above the true value In the absence of 
disease. 



Bilirubin (serum) 



l'B below 0.25 mg.% 
TB below 1.50 mg.% 



Hemolytic jaundice - Total bilirubin Is in- 
creased but the fraction reacting promptly 
(1' B) is not. 

Obstructive and Hepato-celiular Jaundice - 
Both fractions are increased. 



Bromide (serum) 



Less than 3 mg. % 
(non-specific color) 



Bromsulfalein Test Below 10% at 30 mia. 

Below 7% at 45 min. 
0-1% at 60 min. 
Clearance 
5. 33^0. 75 ml. /min. /kg. 

Calcium (serum) 9 to 11. 5 mg.% 



Increased In Bromide Intoxication, clinical 
signs of which are usually evident at serum 
levels of 100-200 mg.%. 

This test is most useful In liver disease 
without jaundice. In advanced cirrhosis 
50% may be retained after 45 minutes. 
This Is one of the most useful liver func- 
tion tests. 

Serum calcium may be altered in the fol- 
lowing ways: Deficient absorption (Vita- 
min D deficiency) usually results in a low 
normal serum calcium. Diseases of the 
parathyroid gland may cause Increases 
up to 20 mg.% in parathyroid exceesj 
decreases (below 6 mg.%)in deficit. 
Serum calcium also varies Inversely 
with serum phosphate (for example 
in terminal uremia) . With phosphate 



305 



Determination 



Normal Range 



Pjgggaglgn of Variations In Disease States 



Calcium (coot.) 






Calcium (urine) 
"Sulkowltch teat" 



Moderate 



retention, calcium will fait). Changes In 
plasma proteins will cause change u :r, total 
serum calcium since part of the calcium 
Is "bound*' to the protein. 

Urine results reflect (ionized) serum cal- 
cium. No precipitate means a total serum 
calcium less than 8.6 mg.%. A fine cloud 
means serum calcium is normal. A 
heavy cloud means a total serum calcium 
of more than 12. 5 mg.%. 



CephaUn Flocculation - see '•Thymol Turbidity" 



Chloride (serum) 



AsHaCl 866-620 

mg.% 
As CI" 340-376 

mg.% 
96-106 mXq. /I. 



For variations in disease see Acid-Base 
Equilibrium (Appendix) . Serum CI" Is 
lowered during excessive loss of Cl~ as 
In diarrhea and vomiting and in some In- 
fectious diseases such as meningitis and 
pneumonia. Increases are rare and poorly 
understood. 



Cholesterol 
Cholesterol 
Caters 



Congo Red Test 



Creatine (urine) 



Creatinine 
(serum) 
(urine) 



128-160 mg.% 
60-T6% eaters 



If ore than 60% 
retention 



0-300 mg. /24 hr. 



0.7-1.4 mg.% 
l-2g./24hr. 



Increases in nephrosis (600-700 mg.%) 
diabetes mellltus, obstructive Jaundice, 
hypothyroidism (600-700 mg.%). Decreas- 
es are seen In hyperthyroidism (60-100 
mg. %) and some other infections, wasting 
diseases and anemias, m liver diseases, 
the % due to cholesterol esters decreases 
(down to 10%). 

This is a test for "amyloid disease". De- 
posits of "amyloid" In tissue absorb congo 
red. m amyloid disease less than 40% of 
the dye will remain in the plasma. In ex- 
treme cases as little as 10% may remain. 

Urine creatine increases in nuclear disor- 
ders of muscle, starvation, fevers, etc., 
especially In muscular dystrophy. 

Serum creatinine Increases In renal di- 
sease, but later than does urea, and more 
than S mg. % in a chronic nephritis 



306 



Determination 



Normal Range 



Discussion of Variations In Disease States 



Creatinine (cont.) 
(serum) (urine) 



Indicates a poor prognosis. Creatinine 
clearances are becoming increasingly 
used for renal function studies. 



Fibrinogen 



0.113-0.380 mg.% 



Glucose 
(blood) 






00-120 mg.% 
60-80 mg. % 



Glucose tolerance 



Icteric Index 



4-6 units 



Flbrinogenopenla (decreased fibrinogen) 
may be congenital or acquired, byjn vitro 
clotting or by severe hepatic dysfunction. 
Increases are seen In most Infectious di- 
seases except typhoid fever. 

Total reducing substances. 
True glucose. Increases are seen in dia- 
betes mellitus up to 500-1000 mg. %. De- 
creases in insulin shock to 20 mg. % or 
lower. 

See the Manual for Interpretation p. 184. 

7-15 units may be measured In latent (sub- 
clinical) Jaundice. Over 15 results In ob- 
vious jaundice. Increases are seen in 
many types of liver and gall bladder and 
bile duct disease as well as in hemolytic 
disorders. Carotenes (pro-vitamins A) 
may contribute to the color of the serum 
as well as the more usual bilirubin (see 
BILIRUBIN), 



Kepler-Power Water Test- See under this heading in Manual, p. 85. 



Lead (urine) 



Sulkowitch Test 

Nitrogen 
Non-Protein 
(Blood NPN) 



30-80 ug/24 hr. 

See CALCIUM (urine) 
25-35 mg.% 



Phosphate (serum) 2 . 5-4 . 6 mg . % 

(adult) 
4.0-6.0 mg.% 



In acute lead poisoning up to 300 ug/day. 
See PORPHYRINS (urine). 



Since NPN Is composed largely of urea, 
creatinine, and uric acid (along with amino 
acids) the same sort of Interpretations 
arise. See under these listings. 

Increases: Chronic nephritis up to 20 mg. % 

Hypoparathyroidism up to 7 mg. % 
Decreases : Hyperparathyroidism to 2 mg. % 



307 



Determination 



Normal Range 



Phosphate (serum) (cont.) 



Phosphatase (serum) 
(Bodansky units) 



1.6-4.0 units/100 ml. 

(adults) 
Up to 12 unlts/100 ml. 

(child) 



Discussion of Variations In Disease States 

Insulin Injection or glucose tolerance test 
to below 3 mg.%. During treatment of dia- 
betic coma the value may fall to below 
1 mg.%. 

In general, serum phosphate and serum 
calcium levelB rise and fall reciprocally. 

Increases are seen during actual or attemp- 
ted bone regeneration. 
Rickets: up to more than 100 units 
Paget 'b Disease of Bone: up to 500 units 
Hyperparathyroidism: 20-700 units 
In liver disease the phosphatase of serum 
may be raised because of failure of the 
normal excretion of this enzyme in-the bile. 



( 



Porphyrins (urine) 
Porphobilinogen 






Negative Found present In acute Idiopathic porphyria 

along with uro -porphyrins (absent in pure 
cutaneous types). 

Ordinary tests are Interpreted as normal- 
ly negative. In the rare congenital porphy- 
ria, large amounts (50-100 mg. /day) are 
excreted, mainly Type L 
Coproporphyria I and HI are normally pres- 
ent in urine. They are Increased In liver 
disease (up to 1 mg. /day), heavy metal poi- 
soning (5-10 mg. /day). 
Porphyrin precursors are present to a variable extent. Porphobilinogen is only one of 
several precursors, whose presence may be demonstrated by appropriate treatment 
(such as heat, light, or LJ of the urine or of urine extracts. 



Uro-porphyrins Less than 10 ug/day 



Copro-porphyrins 50-250 ug/day 



Sodium (serum) 
Potassium (serum) 



134-144 mEq. A 

3.5-5.3 mEq./l 



Protein (serum) 



Total 6.5-7.6 g.% 
Albumin 4. 0-5. 5 g. % 
Globulin 1.7-3.0 g.% 
A/G ratio 1.4-3.0 



See "Acid-Base" Equilibrium (Appendix). 
In general excessive loss of body fluids 
decreases serum Na, which is also low In 
Addison's disease; an increase In serum K 
is seen In Addison's disease; a decrease 
in treated diabetic coma, diarrhea and 
vomiting. 

Albumin: an increase in albumin occurs 
only In dehydration and the rise Is paral- 
leled by a rise in globulin; thus the A/G 
ratio is constant. A decrease in albumin 



( 



308 



Determination 



Normal Range 



DlscuaBl on of V ar iations tn Diseas e States 



Protein (cont.) 






Prothrombin Time 11-16 seconds 



Salicylic Acid 






O-lmf.% 
(non-specific color) 



la Been in (a) loss of albumin in urine; (b) 
decrease*! formation In the liver; (c) in- 
creased metabolism of protein; (d) protein 
starvation; (e) diversion of protein syn- 
thesis toward globulin. Globulin 1b In- 
creased in liver disease, multiple mye- 
loma, and in chronic infections. The al- 
bumin Is often reciprocally reduced, re- 
sulting In a A/G ratio below 1 (a "re- 
versed" ratio). For fibrinogen, see above. 
For gamma globulin see zinc turbidity. 

For discussion of the clotting factors see 
p. 324. A prolonged prothrombin time 
(lowered percentage) is seen in liver 
disease, vitamin K deficiency, dicoumarol 
treatment of poisoning. 

In the treatment of rheumatic disease, 
levels of 35 mg. % may be desired. This 
borders on the toxic level, so close lab- 
oratory control may be required. 



Sulfonamides 



None 



The ','therapejutic level" varies but usually 
1b between 10 and 15 mg. % but In intra- 
cerebral Infections some workers have 
uBed levels of 30-40 mg.%. 



Thiocyanate 



None 



Thiocyanate may be used for treatment of 
hypertension or for estimation of "total" 
extracellular fluid volume. Toxic levels 
are reached at about 12 mg. % In serum. 



Thymol Turbidity 

Zinc Turbidity 
Cephalin 

Flocculation 



-4 units 
2-8 units 

0-1 units 



These three tests roughly parallel each 
other and depend on (a) increased IT-glob- 
ulln; (b) decreased serum albumin; (c) qual- 
itative changes in serum globulin, serum 
albumin, and phospholipids. Diseases In 
which these tests are usually positive are: 
Infectious hepatitis, cirrhosis, Infectious 
diseases with antibody production (gamma- 
globulin) . 






309 



Determination 



Normal Range 



Discussion of Variations In Disease States 



( 



Urea Nitrogen 
Urea Clearance 



8-18 mg.% 

75-126 % of average 

normal 
54 ml./mtn.=C 8 
75 ml. /mtn. *C m 



Blood urea nitrogen (BUN) Is Increased In: 
(a) pre-renal states, e.g. , dehydration 
with reduced plasma volume and thus de- 
creased glomerular filtration rate; (b) re- 
nal states, e.g. , acute glomerulonephri- 
tis, mercurial poisoning, etc. ; (c) poet- 
renal states, e.g., obstructive lesion of 
ureter. 

Decreases In BUN may be seen in very 
severe hepatic failure due to failure of the 
liver to deamin&te amino acids. 
Urea clearance is a more sensitive indica- 
tor of renal disease than Is the BUN. 



Uric Acid (serum) 2-6 mg . % 
Uric acid clearance 6 - 18 ml. /mtn. 



Increases in serum uric acid are seen in: 
(a) renal disease 4-20 mg.%; (b) gout 6 to 
10 mg.%; (c) leukemia up to 10 mg.%; (d) 
toxemia of pregnancy up to 20 mg. %, 
Uric acid clearance averages about 10% of 
the urea clearance, and Is being used to 
Investigate pre-eclamptlc states. 



( 



Zinc Turbidity 



See "Thymol Turbidity," above. 



URINE EXAMINATION 



Chemical Tests: 
(a) b/ concentration 



24 hr. - pH 6,0 Usually (but not always) the urine will tend 
Random 4. 6-8. 2 to be alkaline In alkalosis and acid In acid- 
dosts. See "Acid-Base Equilibrium" 



(b) Specific 
gravity 



24 hr. 1.016-1.026 
Random 1-602 - 1.040 



Specific gravity varies with the balance be- 
tween Intake of fluid and loss by the other 
routes of water disposal (perspiration, vom- 
iting, diarrhea, etc.). m diabetes insipidus, 
very large volumes of very dilute urine are 
excreted. In diabetes meilltus, gtucose ex- 
cretion may greatly increase the specific 
gravity, due to glycosuria. 



310 






Dcte rtnl nation 



Normal Range 



Dlncunnlon of Varlntlonn In Diucane States 



Chemical Tests (cont.): 
(c) Protein Qualitatively 

negative 



Several types of proteins may be found In 
urine: (a) albumin and globulin; (b) fibrin- 
ogen; (c) gross blood; (d) abnormal pro- 
teins - such as "Bence-Jones. " Pathologi- 
cally the protein may be due to (a) pre- 
renal causes such as dehydration, Intra- 
abdominal pressure, fevers, etc.; (b) 
renal - all types of renal disease may 
cause proteinuria such as glomerulone- 
phritis, nephrosis, etc.; (c) post-renal - 
addition of protein to urine after It leaves 
the tubules may occur from the renal 
pelvis on; such as ureter, bladder, ure- 
thra, etc. ; (d) abnormal proteins -e.g., 
"Bence-Jones" protein may be excreted 
through the glomerulus. 



(d) Reducing Substances 

"Glucose" Less than 0.1% 



Glucose is found In urine of all persons 
when their blood sugar is elevated above 
the "renal threshold" such as in (a) dia- 
betes mellltus; (b) hyperthyroldismjpltul- 
tarlstn, or-adrenalism; (c) severe liver 
or pancreatic disease; (d) infection; (e) an- 
esthesia or asphyxia; (f) renal glycosuria- 
low renal threshold as in pregnancy. May 
be found in urine during lactation. S may 
be very important to determine the exact 
identity of the sugar. 



(e) Acetone Bodies Negative 

Acetone 

Aceto-acetic acid 
B-hydroxy-butyric acid 

(f) Bile Pigments Negative 



These substances are present in acidosis 
due to excessive fat metabolism as in un- 
treated diabetes mellltus, and starvation. 



Bilirubin is found in the urine in cases of 
obstructive Jaundice and most cases of 
hepatocellular jaundice. It la not present 
In most cases of hemolytic Jaundice. Urine 
levels may be elevated as early as nine 
days before clinical Jaundice In Infectious 
hepatitis. 



311 



lX'tormliiiiMon 



Normal Range 



DlHcuHriion of Variations In Dluease States 



Urine Kxamlnatlon (cont,): 
(n) Occult lllcod Negative 

"llcn/.ldlne Test" 
"Tolltlinc Teat" 
"Giuilac Teat" 






If the hemoglobin la present aa rbc micro- 
scopic examination la more sensitive. 
However, this teBt la also used to verify 
hemoglobinuria. 



SPINAL FLUID EXAMINATION 



Xanthochromia 






A yellow color usually due to hemoglobin 
from destroyed rbc. 



Turbidity 



Glucose 



Chlorides 



Proteins 









50-80 mg.% 



May be due to bacteria or polycythemia 
(pus). 

Decreased In bacterial meningitis and 
hypoglycemia. Increased In hyperglyce- 
mia. 









700-760 mg.% NaCl Decreased In bacterial (especially tuber- 
culous) meningitis. 



15-40 mg.% 



The protein la matnly albumin. Increased 
in meningitis, polyneuritis and tumors of 
the C.N.S. It may be of Importance to 
differentiate albumin from globulin. For 
this technique see reference texts. 



( 






























312 



SAMPLE UEQUMED FOH AHALY3UJ 



Determination Be 


rum needed 


Blood needed 


Anticoagulant 




ml. 


ml. 




Acetone 


1 


5 


none 


Alcohol, etiiyl 


2* 


5* 


oxalate 


Alcohol, methyl 


4* 


5+ 


oxalate 


Amino Acid N 


1 


5 


none 


Amylase 


4 (0.4) 


10 (2. 0) 


none or oxalate 


Ascorbic acid 


5 


16 


none 


Bilirubin 


4 


10 


none 


Bilirubin, micro 


0.2 


1-2 


none 


Bromide 


4 


10 


none 


Bromsulfaleln 


1 


6 


none 


Calcium 


5 


10 


none 


Carbon Dioxide 


2*** 


8*** 


oxalate 


Carbon Monoxide 


0.4* 


5* 


oxalate 


Cephalln Flocculatlon 


0.4 


6 


none 


Chloride 


0.4 


5 


none 


Cholesterol 


4 (0.2) 


10(1) 


none 


Congo Red 


2 


5 


none 


Creatinine 


4(7) 


10 (15) 


none 


Esterase 


4 


10 


none 


Fibrinogen 


3* (1)* 


5* 


oxalate 


Glucose 


2+ 


5* 


oxalate 


Icterus Index 


1 


6 


none 


Iodine (protein bound) 


2 


5 


none 


Iron 


8 


20 


none 


Iron-binding capacity 


12 


25 


none 


Lipase 


4 


10 


none 


Phosphate, Inorganic 


2 


6 


none 


Phosphatase 


2 (0.2) 


5 (1) 


none 


Potassium 


1(0.4) 


5(1) 


none 


Protein-total 


1(0.2) 


5(1) 


none 


A/G ratio 


1 


5 


none 


Prothrombin time 


0.2** 


4.5** 


oxalate 


Prothrombin consumption . 2*** 


5*** 


none 


Salicylates 


1 


5 


none 


Sodium 


1 (0.4) 


5(1) 


none 


Sulfonamides 


1* 


5* 


oxalate 


Thlocyanates 


2 


5 


none 



313 



Determination 



Scrum needed 
ml. 



Blood needed 
ml, 



Anticoagulant 



( 



Thymol turbidity 
Transaminase 
Urea 

Uric Acid 
Zinc turbidity 



0.2 

0.6 

2* (0.4) 

2 

0.4 



6 (1) 
5 

5*(1) 
5 

5 



none 

none 

oxalate 

none 

none 



The above volumes represent the volume of serum (or other fluid) needed for 
the actual analysis in duplicate; the volume of blood which is required for the 
production of that amount of serum with a margin of safety; and the type of 
anticoagulant required. 

♦Whole Blood **Speclal oxalate required ***Special treatment required 



314 



CONTROL PROCEDURES IN THE CLINICAL CHEMISTRY LABORATORY 



The clinical laboratory should provide itself with a system of control which will 
alert the analyst so that errors will be quickly detected. Some of these systems are 
listed below. 

A. Analysis of known solutions: 

In most of the methods listed, the use of standard solutions Is part of the pro- 
cedure outlined and Is therefore an Integral part of the system of control. Another 
part of this method, when the photometric technique is used, is the maintenance of 
suitable records, so that any untoward variations in the amount of color produced by 
the standard (and by the blanks} will at once cause an investigation of the cause. 

B. Replicate determinations: 

This is the only procedure which will allow an estimate of precision (see statis- 
tics, p. 317). m most cases the replication should start at the original source material 
bo as to Include all the possible variations in the measurements which are made. In 
most clinical chemistry laboratories, single analyses only are made . It would be better 
If all analyses were done at least in duplicate . 

C. "Blind analysis: " 

Solutions of pure compounds, whose composition is known only to the laboratory 
director and not to the analyst c an be a very convenient method of control. If a number 
of such solutions are prepared, each suitable for several different determinations with 
different known amounts of substances present, this will prevent conscious or uncon- 
scious bias in the reported results. 

D. Serum Pool Analysis: 

The "known" solution may be a serum pool. A large volume of serum may be 
pooled, divided into convenient portions and frozen. Then, each day or week, one 
sample may be thawed and analyzed. This method is limited to the control determin- 
ations of those substances not altered by this freezing technique. Commercial prep- 
arations are available which have been dried from such a frozen state and when re- 
constituted with water, serve as a very convenient 'standard serum pool. A number 
of such pools may be collected, analyzed and used for control purposes as outlined 
under C_ above. 

E. Laboratory inter-comparison : 

It Is extremely difficult to make comparisons between laboratories due to the 
use of different procedures and due to the difficulty of transporting specimens without 
chemical alterations. This, Is, however, potentially a very important method of con- 
trol. It Is especially convenient for two or three laboratories to serve as a continual 
check on each other by a system of continuous sample exchange. 



315 



F. Hartfit; of variation: 

Tlie portion writing reports and checking results should be familiar with the 
usual range v( values obtained in each test, not only for normal cases, but also In var- 
ious pathological variations. Whenever the results arc outside the realm of possibility 
or even probability, the results should be rechcckcd. In this judgment process, the 
diagnosis is important, so the request card should be consulted (even though frequent- 
ly Its information will be Inadequate). Also, the results of other tests on the same 
patient bearing on the same problem will help, 

1. Normal Ranges 



Most laboratories maintain a list of normal values. These are usually ex- 
pressed as a range. Values outside this range are regarded as abnormal or at least 
with suspicion that they may be abnormal. In order to more clearly comprehend what 
is meant by "variation" and "normal range" we list the types of variability encountered 
in the field of clinical chemistry. 

a . Analytical variation. This is the range of values to be expected if a large 
number of identical samples of the same serum are carried through the analytical 
steps involved. 

b. Individual variation . This Is the range of values to be expected If the serum 
(for example) of a large number of different persons is analyzed. The samples are 
taken at comparable times to minimize the effect of certain variables mentioned In the 
next paragraph. 

c . Physiological variation: This Is the range of values to be expected If samples 
are taken from one Individual at various times during the day or night} at various times 
during his life; or under various types and degrees of physiological stress . Some of 
these variations are predictable or controllable; others are uncontrollable. They are 
due to such variables as: age, Bex, previous dietary history, body weight and height, 
mental state, degree of muscle activity, etc. 

To minimize some of these variations blood samples are usually taken with the 
patient in a "fasting" or "post-prandial" state, some 12-14 hours after the last meal. 
In some cases it is important to avoid exercise previous to blood collection, or during 
a series of blood collections. 

Few of the determinations in this manual have been studied sufficiently to obtain 
a complete knowledge of the effect of all of the factors listed above, fa most of them, 
the analytical precision can be stated, and in most, the individual variation of the fast- 
ing samples Is known but the physiological variation Is for the most part still incom- 
pletely studied. 



. 



316 






STATISTICS 



Statistical analysis 1b a vast and growing field with many facets. It will suffice for 
our purposes to outline very simple concepts of two properties of collected data. These 
two properties may be termed measures of 

a. The central tendency 

b. The dispersion or the "spread" of the data. 

The average or arithmetic mean will be used to express the^ central tendency. This 
Is merely the sum of all the values determined, divided by the number of the determin- 
ations. 

The tendency to dispersion Is less easily defined. In any large series of analyses 
It will be noted that the data may be expressed somewhat as shown In the following 
bar graph. 

10 r 



a 



NO. OF 
VALUES 



tllfl 



/ 



V- 



/ 




\ 



CONCENTRATION 



s 



^ 



X 



X 



EC 



A* 



The bar graph shows the actual results of a series of analyses, on normal serum, 
while the smooth solid curve is a theoretical curve based on, an Infinite number of 
analyses. 

Dispersion of the results obtained In a series of analysed (say on normal serum) 
can be expressed as a range from the lowest to the highest values. This Is perhaps the 
most common method used in clinical pathology today. It Is better, however, to calcu- 
late the standard deviation by formulae (1) or <2). 

The standard deviation Is a mathematical measure of the dispersion or "spread" 
of the data to either side of the mean or average value. K may be calculated using 
formula (1). 



31T 



7 »-* 



( 



(') <^ =1/- M r. " J * = arithmetic mean of N determinations 

x s value of a single determination 



cr ;: standard deviation x - x = deviation of a single determination 

jjr = "sum of" from the mean 

M a mean or average 

Thus, after a method Is well-known and the mean and the standard deviation have 
been established, a value which deviates from the mean by more than 3 times the stand- 
ard deviation (3o-) can be considered "abnormal" or "not due to random errors" since 
only 3 tlmeB in a thousand (0.3%) will the deviation exceed 3*-by mere chance. 

When this Is done, we can say with considerable certainty that 68% of all normal 
values will fall In the range between the mean minus one standard deviation and the 
mean plus one standard deviation (M r_ i<r) ; that Is to say, that the range A to A 1 in the 
figure Includes about 68% of the area under the distribution curve. 

If we extend the range to (M / 2a) then 05% of all normal values will be included; 
(M i 3a) Includes 99. 7% of all normal values. 

Another method of calculation of the standard deviation based on duplicate deter- 
minations is given in formula (2). 

(2) <r =y < x i " x 2> 

f N - 1 

where Xj Is one value and x 2 is the other value of a pair of determinations on 
the same sample. 

Example : Two different technicians analyzed the same series of serums for total 
cholesterol and collected the following data. 



318 



Scrum 


CKolcBterol 


(mg.%) 


f 


Value #1 


Value #2 


32 


97 


95 


33 


104 


108 


35 


111 


108 


39 


104 


108 


40 


135 


123 


41 


111 


108 


42 


111 


95 


48 


97 


95 


50 


118 


108 


53 


104 


101 


54 


U8 


123 


58 


111 


115 


59 


97 


101 


70 


97 


115 


73 


126 


123 


75 


126 


129 


880 


135 


126 


885 


126 


118 


887 


135 


117 



(i-2) a-2) 2 



2 4 
4 16 

3 9 

4 16 
12 144 

3 9 The data are 

16 256 

2 4 analyzed 
10 100 

3 9 using formula 

5 25 

4 16 (2) 
4 16 

18 324 
3 9 

3 9 

9 81 

8 64 

1435 =2,(xi - x 2 r 

18 r N-l 



VT435 JT^ 



79.7 - 8.92 



The standard deviation la thus 8.92 mg. cholesterol per 100 ml. serum. A dif- 
ference, then, of more than 26. 8 mg. (3 x 8. 92) between two analyses on the same 
serum would occur only 3 times out of a thousand analyses and thus probably repre- 
sents something other than random error. 

This same technlc applied to serum samples from a number of normal human 
beings would allow us to state that a variation from the mean or average normal 
value which exceeds more than 3 times the standard deviation represents more than 
random variation and may be due to physiological or pathological variation in the 
patient. 

Sometimes it is convenient to express the standard deviation in terms of "% of 
the mean. " It is then called the "coefficient of variation. " 

Example: If a series of determinations gave a mean and standard deviation of 
124 £ 8.9, then the coefficient of variation would be 

JbJ* x 100 = 7.2% 
124 

319 






ACID BASE EQUILIHUIUM AND WATER BALANCE 

The cations (or "base") Na^ and k/, and tlic anions CI - and HCO ~ play an import- 
ant role, by means of their effective osmotic pressure, in the regulation of the exchange 
of water between the cells of the body and the plasma and Interstitial fluid. They are 
also concerned with the acid-base equilibrium of the body. 

It is Impossible to discuss the changes in any one of these Ions without discussing 
the others because of the close interplay between and among them. As a whole, the body 
strives to maintain (a) a constant electrolyte composition (especially In regard to total 
osmotic pressure, which is almost equivalent to saying total electrolyte concentration) 
and (b) a constant pll. 

The body is continually manufacturing "volatile" acid carbon dioxide (H„CO,), which 
Is excreted largely through the lungs. In smaller quantities. It forms HC1, H_SO. , 
H3PO4 and others. Most of these latter are non-volatile and must be excreted by the 
kidney. It is the momentous problem of the lungs and kidneys selectively and judiciously 
to excrete the correct substances In the right amounts, so as to maintain the two pre- 
viously mentioned Important quantities, (a) osmotic pressure or electrolyte concentra- 
tion and (b) pit. 

The pH depends on the ratio of HCO ~/H 2 C0 3 and changes In the acid base equilibria 
of the body arc quickly reflected In the HC0 3 ~ of the plasma. The H 2 co 3 of the plasma 
will rise and fall with the HC0 3 ~ of the plasma and the ratio HCO3 /H 2 CO a will be very 
close to 20/1; this control Is maintained by variation In the depth and rapidity of respira- 
tion and Illustrates the strong attempt by the body to maintain a constant pH. HC0 3 ~ then 
is the most labile or changeable of the electrolyte components and any change In the 
ethers will be reflected In a change In HCO,", Thus: 

1. If Na^ is decreased; HC0 3 ~ is decreased. 

2. If Cl~ is decreased: HCO3 - Is Increased. 

3. If CI" Is increased: HCO- - Is decreased. 

4. H non-volatile anions such as SO|, POf (renal failure) or acetone body acids 
such as aceto-acetate or B-OH butyrate (diabetes) are Increased, then HCO^ be- 
comes decreased. 

The above relationships of course depend on two axioms: 

1. The total cation concentration must be equal to the total anion concentration. 

2. In general, the total electrolyte concentration is kept relatively constant. 

The inter-relationships may be Been more clearly by a few examples, keeping In 
mind that the kidney also operates In the regulation of acid-base equilibrium by (1) ex- 
creting an acid urine, and (2) synthesizing ammonia to replace Nar as a cation. Both of 
these methods conserve body base (Na 7 ;. In renal failure inadequacy of these "base- 
conBervatlon" mechanisms causes a loss of Nar into the urine and a decrease In plasma 
Na' concentration. 

Severe loss of plasma volume also may cause a secondary type of renal insufficiency 
or failure with similar results. 

321 



EXAMPLES OF ELECTROLYTE CHANCES IN DISEASE 

Norma l valuta meq. /liter Average 

Na 137-147 142 

Cations K 4. 1-6.G 5 

Ca 4.1-5.6 5 

HCO „" 23 - 31 27 

Anio ' 13 CI" 3 96-105 103 



I. Severe Extracellular Fluid Lobh "Dehydration" : This may be caused by inability to 
eat or deprivation of water and food. No changes are seen In the concentration of 
electrolytes at first. The plasma volume decreases, and the electrolyte components 
of plasma are excreted in the urine proportionately to the plasma volume decrease. 
When the plasma volume has decreased so that renal failure occurs, loss of Na^ 
Increases, serum Na^ decreases, non-volatile anions Increase and HCO^ decreases sec- 
ondarily to the changes In Na** and non-volatile anions. 

n. Vomiting (Severe) : Decreased plasma Cl - due to loss of Cl~ in HC1 of gastric juice. 
There is a decrease in sodium and Increase in non- volatile anions because of de- 
creased renal function secondary to reduced plasma volume. (In mild vomiting only 
a decrease In Cl" and an Increase in HCOg" may be seen.) 

m. Chronic Nephritis (Advanced): The kidney's reduced ability to excrete fixed non- 
volatile anions results in 

a. An increase of these anions In the plasma. 

b. A decreased plasma HCO3"*. 

c. The reduced ability to conserve base results in a reduced Berum Na^. 

IV. Diarrhea : There is a decrease in Na'' because of loon of secretion high in Na'* 

content. A further decrease in Na^.and an increase In non-volatile anions and thus 
a decrease in HCOo" is seen in more severe diarrhea. 

V. Addison's Disease: 



A. Primary defect: a deficit of adrenal cortical hormones results in urin ar y 
loss of Na/ and retention of KX . 

B. Results in 

1. Decreased serum Na^ (and decreased plasma volume), 

2. Decreased HC0 3 ~ (parallels Na^ decrease). 

VI. Diabetes : 

A. Defective metabolism of carbohydrates which results in an excessive cata- 
bolism of fats and a hyperglycemia. 

B. The by-products of fatty acid metabolism, B-OH butyric acid, and accto- 
acetic acids are strong acids which require the excretion. of much Na* 1 and 

322 



result In a lowered plasma Ka*". Also glucose Is excreted In the urine, 
requiring the excretion of waler and some Na>* thua further lowering 
plasma Ka . 

C. Plasma Na^ haa decreased, non-volatile anions have increased; therefore, 
HC0 3 ~ decreases to maintain anion-catlon equivalency. Since H„CO 
must then be reduced to maintain the pH constant, hyperpnea is clinically 
observed. 

D. As the "dehydration" proceeds due to loss of water along with glucose, and 
with the excretion of the sodium salts, renal dysfunction occurs which re- 
sults in still further loss of Nar , and further increase in plasma non- 
volatile anions. 

E. ThlB is a "vicious cycle" and finally the pH Is lowered, and the plasma vol- 
ume may be lowered enough to cause "shock" or peripheral vascular collapse 
with a low blood pressure. 

Laboratory Tests: 

Blood - Hgb & hematocrit - both high - suggesting decreased plasma volume - 

he moc once nt ration. 
Plasma - Na^ - low 

HC0 3 ~- low 

CI - - normal or low 

pH - low 
Urine - Sugar - positive - glycosuria 

Acetone bodies - positive 

During and after vigorous treatment of diabetic coma, potassium Is rapidly returned 
to the cells from which it was drawn or lost during the acidosis. Therefore it is import- 
ant to replace K' and to control Its use therapeutically by plasma K^ determinations. 

Following are the normal values and conversion factors for the various Ions In serum: 
Convers ion Factor Normal Range Average 

Sodium (mg. /100 ml.) x 10 = mEq. Na^/l. 13T-14T mEq. /h 142 

23 
Potassium- yng./lQO ml) x 10 = mEq. K r /L 4.1-6.6 " 6 

39 
Calcium - (mg./lOO mljx 2 xlO _ m Eq. Ca^/1. 4. 1 - 5. 6 " 5 

40 
Bicarbonate - volumes % - mEq. HCO3/I. 23 - 31 " 27 

2.24 
Chloride - <mg. /100 ml.) x 10 = mEq. Cl~/1. 96 - 106 f 103 

(expressed as 58 . 45 

mgm. of NaCl/100 ml.) 

Protein (anion) (g./lOO ml.) x 2.43 = mEq. Prot.'/l. 15-19 M 17 

POI 1 - 8 (mg./lOO ml.) x 0.580 = mEq. PO^ 1 * 8 /!. 17 - 2.9 2.3 



323 



COAGULATION OF DLOOD 



In order to understand (1) the purl prothrombin plays In tlic coagulation of blood, 
(2) Its Importance to the practice of clinical medicine, and (!)) the importance of tlic 
various factors and reagents Involved in the laboratory determination, an abbreviated 
explanation of tlic clotting mechanism will be presented. 

The processes involved in the coagulation of blood may be divided into three steps (for 
purposes of discussion). These are: (1) Activation of thromboplastin , (2) Conversion 
of prothrombin to thrombin, (3) Conversion of fibrinogen to fibrin. 

Stage I: Activation of thromboplastin. A number of factors are important in this acti- 
vation: 

A. Platelet factors 

B. Anti -hemophilic factors 

i. Anti -hemophilic globulin (AHG) in plasma, not present In aged serum, 

not adsorbed by BaSO^. 
ii. Plasma thromboplastin component (PTC) ("Christmas factor") present 

in serum, adsorbed by BaS0 4 . 
111. Plasma thromboplastin antecedent (PTA), present in aged serum, not 
adsorbed by BaSO^. 

C. "Contact factor" — wettable surfaces increase rate of formation of thrombo- 
plastin. 

D. "Tissue factor" — tissue extracts increase rate of formation of thromboplastin. 

E. Ca^r is necessary for thromboplastin formation. 

There is disagreement as to the actual Bource of the thromboplastin. It may be present 
in plasma as an Inactive precursor (Quick's thromboplastlnogen) , and merely activated 
by the tissue and platelet factors. Others believe the reverse is true, that the inactive 
precursors are present in tissue or in platelets. 

Stage 2: Conversion of prothrombin to thrombin. Again a number of different factors 
are important in thiB conversion. 

A. Thromboplastin - derived from Stage 1. 

B. Ca' ^ is also necessary in Oils stage. 

C. Prothrombin Is present in plasma and Is converted to thrombin. It is relative- 
ly stable, and is adsorbed by BaSO*. 

D. Accessory factors 

1. Proconvertin (plasma) or convertin (serum) Is required for the conversion 
of prothrombin to thrombin (physiologically),. It is stable, and is adsorbed 
(as Is prothrombin) by BaS0 4 - 
ii. Proaccelerln (plasma) or accelerin (serum) merely accelerates the con- 
version of prothrombin to thrombin, (I.e. , is not absolutely required) 
in the physiological system. It Is quite labile and disappears rapidly from 
37° C. incubated plasma, it is not adsorbed by BaSO^ (differing from 
proconvertin and prothrombin). 

324 









OWREN'S (l r J53) THEORY OF BLOOD COAGULATION 



Reference: American Journal of Medicine 14, 201 (1953), 



This schema represents the Inter-re latlonshJps between the various stages of, 
coagulation. It should be noted that the first stage of coagulation Is under Intensive 
Investigation now and at least two more factors, present In plasma have been shown 
to be necessary for the rapid formation of thromboplastin In blood. TheBe are plasma 
thromboplastin component (PTC)* the "Christmas" factor; and plasma thromboplastin 
antecedent (PTA)*. There may be more. 







tj 


T1 




JO 


°m 




o 


«^ 




H 


m? 




I 


i 




JO 

o 

03 

z 



TJ 












o 


oo 

Z 1 

* 




T^jaz 

>o-* 
o> 2T 




tn 

c 




o 

z 
-i 
> 


JO 




h| ' 




m 




o 


-1 




zo 




to 




—i 


z 




i 












y 










*s ^^-— ^^ 




"OH 






>J0 














mo 






-i2 






— CD 






1 














324a 



( 



( 



Sta ff e 3: Conversion of fibrinogen to fibrin (clot). 

A. Throrabin-produced in Stage 2. 

B. Fibrinogen — needed as substrate to the thrombin action. No other factors 
are required. 

The characteristic which above all else has been most Intriguing to investigators of 
coagulation is the suddenness of the clotting once it started to occur. This rapidity Is 
due to what electronic engineers call "positive feed-back", or what biologists call 
"auto-catalysis", that Is, In the various stages. of coagulation, certain products stim- 
ulate or accelerate the primary reactions, thus, further accelerating the overall 
process. This can be seen by a view of Owren's (1963) schema seen on the facing 
page, American J. Med. XIV, 201(1953). 






326 






hi addition to these "positive feed-back" uyHtems these aluo arc uoine "negative feed- 
back" mechnnimmt or inhibitors system. 

A. Accclerln > inactive accclerln. (catalysed by thrombin) 

B. Thrombin / Ant 1 -thro nib in — y inactive thrombin. 

C. Proflbrinolysin — » fibrlnolysln (catalyzed by fibrlnokinase) 

Fibrin — > fibrin Bplit products {catalyzed by flbrlnolysin) 

D. Thromboplastins are neutralized by antl-thromboplastinB. 

E. Flbrlnolysin is neutralized by anti-fibrlnolysln. 

Sy nonymy: Since there have been many different workers in the field, there have been 
may terms Invented which apply to the same factor. Some of these which may cause 
confusion are listed here: 

1. Pro-accelerin (Plasma) and acceierln (serum) Factor V. (Owren) 
Ac (Accelerator) globulin 

Prothrombin accelerator 

Plasmatic cofactor 

Labile factor - Quick 

Prothrombin A Quick— 1943 

Plasma prothrombin conversion factor (PPCF) 

2. Pro-convertiii (plasma) convertia (serum) 
Serum prothrombin conversion accelerator (SPCA) 
Stable Prothrombin conversion factor 

Factor vn (KoUer) 
Stable Factor 



COAGULATION DEFECTS 

3tage_l: Defects 

Hemophilia: A deficiency of anti -hemophilic globulin of plasma (AKG). 

Thrombocytopenia: A deficiency of platelets and platelet factor. 

"Christmas" disease: A deficiency of plasma thromboplastin component (PTC). 

Named after the family in which this defect was first noted. 
(PTA) deficiency: Described by Rosenthal (1955)* 

* A New First-stage Clotting Component. "Plasma Thromboplastin Antecedent". 
Rosenthal, Blood 10, 120 (1955). 

Stage 2: Defects 

Prothrombin deficiency: due to: (1) Vitamin K deficiency and gross liver disease. 
Vitamin K Is required by the liver before synthesis of prothrombin can be car- 
ried out. Since Vitamin K Is fat -soluble, Its absorption from the Intestine is 
markedly reduced in the absence of biliary and pancreatic secretions. Gross 
liver disease such as cirrhosis and acute yellow atrophy and acute chloroform 
polnoning will interfere with the formation of prothrombin even in the presence 
of adequate dietary or Intramuscular synthetic Vitamin K., 

32G 






('/) Ilfiiir»rrli;i|',lf disease ol lite new born heloti! llws routine pre-natal use of 
Vitamin K wan laiily common (1/400), filiiee lire use of llm Vllamln It In much 
li-nn common, and Ihfifi apparently w;mi ilun In prothrombin deficiency seconds 
» ry In ;i Vllaeiiln K deficiency which Id liirn was probably due In tli»* absence of 
nil adult type of microbial Mora In the. lidunt's liilcollne. In adults, much of the 
Vllii tjilit K required In obtained through lln; kind services of Intestinal bacteria. 
However, Hm 1 exacl coagulation defect In the new-born has not been InvciiMgu- 
led In Hit 1 : ll|',lil of I he newer Btage 'I factors, 

(II) Dleoumarol |ioliionlis|>: Thin chemical causes a clotting defect first nolud 
In cattle feeding on spoiled sweet clover. II If* duo lo a luck of prothrombin 
(mid alno pro-converlln). This flr*l|; and 11k chemical relatives arc used widely 
for the treatment of patients with thrombosis or thrombotic tendencies. 
(')) Congenital Prothrombin Deficiency: Though there have been alleged CUHCH 
In which familial prothrombin deficiency wan present, all but one of Uichc prob- 
Itahly were caiien of proconvertin or pro-accelerin 'deficiency, hcc below. In 
any case, Instances of Congenital failure of the liver to manufacture prothrom- 
bin must hi: very rare, In the presence of adequate Vitamin K and normal liver 
functions. 

Pro-converlln deficiency: (1) Vitamin K deficiency and/or obHlructlve jaundice 
nntl steatorrhea (poor absorption) cause Home decrease In pro-conycrtln. 
(?.) Dlcoumarol as used In treatment of UiromboHJH decreases pro-converlln as 
well ait prothrombin . 

(;i) Congenital — there are id leant six cases of a hemorrhagic tendency from 
early childhood which have born eihown to be due to a pro-convcrtln deficiency. 
(4) Trmnexan treatment: It affects prothrombin very Utile and its main effect 
In lo deeroano pro-converlln, 

J^ni-accelerln deficiency: (1) Liver disease which affects the liver cells decreases 
pro acre Jerln, Obstructive Jaundice (uncomplicated) docs not affect pro- 
uccelerln. 

{2) Posl-opcrallvely pro-accelerln decreases lo a minimum at about the third 
day post-operallvely and returns to normal on about the ninth day, 
(:i) Congenital and familial: a number of cases have been described, especial- 
ly In patients being treated with a nil-coagulants. When there is significant 
time lapne between collections and Hie prothrombin assay, there exists the 
possibility of a Iohh of nro-aeeelerln. 

(4) Coagulation: The process of clotting apparently uses up pro-accelerln and 
In massive In vivo clotting pro-siceelcrln deficiency may occur. 

Klage 3: l»eferls 

I'lbilnogen deficiency: Plasma fibrinogen must be beloW 100 mp,% to affect the 
prothrombin lime. (I) Congenital— -very rare: The patient's bleeding time 
may be nearly normal Illustrating Unit mechanisms other than coagulation are 
Important In control of slight hemorrhage. 
(V.) Massive In vivo elolllng may eaiiHe rapid duflbiinogenalloii of the blood. 



:vn 



Other Coagulation DelcctB : 

"Circulating anti-coagulants" may Interfere with thromboplastin or prothrombin 
or with thrombin activity. Some (heparin) have been shown to interfere with pro- 
accelerin, "Fibrlnolysln" rapidly lyses clots and when present m vivo in large amounts 
may render the blood "Incoagulable", e.g. , In massive Intravascular clotting such as 
post-partum uterine hemorrhage. 

Anticoagulant Therap y: 

Heparin Group : Heparin is a complex polysaccharide with many sulphuric acid 
groups. It Is not effective by mouth but lengthens clotting time when given Intra- 
venously or Intra -muscu la rly. Us duration of action is relatively short (4-6 
hours) and is quite expensive. Its administration Is controlled by estimation 
of whole blood clotting times. Heparin may affect thromboplastin generation, 
prothrombin conversion and pro-accelerln activity. 
Dlcoumarln Group : These drugs affect factors mainly In Stage 2, the prothrombin 
conversion to thrombin. 

Dlcoumarol : decreases prothrombin and pro-convertln; does not affect pro- 
accelerln except perhaps to Increase Its lability. 
Tromexan : decreases pro-convertln, does not affect prothrombin. A short 

acting drug (12-24 hours). 
Hedulln: Apparently affects only prothrombin, but Is a shorter acting drug 
than dlcoumarol. 



References : 

The Coagulation of Blood-Methods of Study by L.M. Tocantina, M.D. , Green 
and Stratton, 1955, 

Human Blood Coagulation. Biggs, and Macfarlane, Blackwell, 1963. 






( 






328 



TURBIDITY STANDARDS 



Re ferences : 

1. Shank. R. E. and Hoaglund, C. L., J. Biol. Chem. 162 , 133 (1946), 

2. Duccl, H. , J. Lab. h Clin. Med., 32, 1266 (1947) 

Some of the methods described depend upon the development of a turbidity by the 
addition of a reagent to Berum, urine or spinal fluid. The degree of turbidity can be 
estimated by the photometer. This Is done in the methods described for thymol turbid- 
ity, gamma globulin, urine and spinal fluid protein. These methods are, in general, 
considerably less accurate than are good chemical methods. 

Originally the unknowns were compared to gelatin standards whose turbidity was 
developed by adding formaldehyde. 

Recently, the use of turbidity standards of barium sulphate suspensions' has become 
popular. These are prepared as described below. 

Reagents : 

1. Barium chloride 0.09G2 M (2%) 

2. Sulfuric acid 0.2 N 

3. Stock suspension 

In a 100 ml. volumetric flask place 3.0 ml. of the 2% BaCL. solution. Cool to 
0°C. in ice bath. Add 0, 2 N H ? S0 4 at 0°C. with shaking up to the mark. Mix rapidly 
and completely. Do not attempt to store. 

4. Dilute Standards - 

20 unit standard 2. 70 ml. suspension 

0.30 ml. H 2 S0 4 



3.00 ml. total volume 

10 unit standard 1.35 ml. suspension 

1.65 ml. H 2 S0 4 
3.00 ml. total volume 

Other dilutions may be used and the amounts may be vdried to give suitable total 
volumes. Place the dilute standards in matched photometer cuvets, read against dis- 
tilled water as a blank, and plot D readings against turbidity units. 

The data can then be used in the preparation of graphs other determinations in 
which a turbidity standard is used. 



329 



Hefliilts of lurbiiUty comparison*! made lining iheoe standards aa given above will be 
expressed In MacLnglan imlla. One Mac Lag tan unit represents 10 mg. protcbi per 100 
ml. of fluid (urlno, spinal fluid, or thymol turbidity, etc.). Recently the A.A.C.C. 
(American Association of Clinical Chemists) published in their first volume of Standard 
Methods of Clinical Chemistry , directions for Shank and Hoagland BaSO, standards. 
The procedure, aa suggested in SMCC, with any given Berum results in twice the num- 
ber of turbidity units as would be the correct number of MacLaglan units. This should 
be considered in comparing results of one laboratory with another. 



CALIBRATION OF PHOTOMETER TUBES 






In order to be able to compare the optical densities of standards, unknowns and 
blanks, it is necessary that the nominal optical thickness of the cuvets or test tubes 
which are used be known or be identical. There are two wayB In which to insure this. 

1. All readings In the photometer are made using the same photometer tube, rins- 
ing with solution each time, and being sure that the tube is oriented identically and is 
clean and dry on the outside at each reading. The cuvet may be fixed in position and 
the solution removed by a drain or by suction. 

2. When it Is Impossible or inconvenient to use Method 1, we must resort to the 
selection of a number of tubes which will give the same effective optical depth when 
used in the photometer. 

Apparatus : 

Photometer using wavelenth 515 mu. 

A stock of test tubes of suitable dimensions. 

The number of test tubes required will usually be about two to three times the num- 
ber of selected tubes finally needed. This number applies only to original selection of 
one homogeneous group from stock supply, since rejects from one group may fit into 
another later group. 

ReaRentB : 

1. Stock solution: Weigh out accurately 0. 2000 g. KmnO, and transfer quantitative- 
ly to a one-liter volumetric flask. Add 1.0 g. KIO^ and 50 ml. syrupy phosphoric acid 
(85-90%). Dissolve completely in distilled water, dilute to one liter, and mix well. . 

2. Diluting solution. Dissolve 1.0 g. of KIO^ in water, add 50 ml. of syrupy phos- 
phoric acid (85-90%) dilute to one liter and mix well. 

3. Working solution for "1 cm." tubes. Place in a one -liter volumetric flask 
150 ml. of stock solution, dilute to mark with diluting solution, and mix. 



330 



t. 



4. Working solution for large photometer tubes ("2 cm. '*). Place In a one- 
liter volumetric flask 100 ml. of stock solution, dilute to mark with diluting solution, 
ami mix. This solution wan developed by Proieswor It. H. Hamilton of Temple Univer- 
sity Medical .School, Philadelphia, Pa. 
Pro cedu re: 

1. Clean the leHl tubcH well by cleaning riuid or detergent and rinse well with tap 
water and then well with distilled water. 

2. Dry in oven (or overnight), Inverted to avoid dust, 
a. Fill 2/3 full of pure distilled water. 

4. Allow photometer to warm up well--one to two hours. Then without Inserting a 
photometer tube, wet the galvanometer at 50.0 (called a center setting) or at some read- 
ing which, with a tube of df Hill led water Inserted will give a reading of 90 to 85. Then 
all the lubes of water are read, checking each time to keep the 0at the predetermined 
value (e.g. , 60.0). Note? If dovc-talllng of the selected tubes with a larger set or with 
other nets la desired It wjll be necessary to choose one selected tube with a certain 
reading wlien filled with distilled water (e.g. , 95% transmittancc) and reserve this tube 
aH a primary standard, protecting It from scratches and dirt, against which all the 
lubcH may he compared. 

Each tube before being read should be Inspected for the following important factors; 

a. CleanllneHB — even a finger print may Berlously affect the reading. 

b. Air hubbies 

c. Orientation in the cuvet holder — the pyrcx trademark is a convenient point to 
UHe. AIho if the tube Is too large and tends to stick, or too loose and tends to wobble. It 
should be diHcardcd. 

d. Striae— any tubes with striae should be discarded. 

5. Hecord all readings on paper, keeping tubes In order. It may be more convenient 
In selecting a short series to record the galvanometer reading on the tube Itself. 

G. Discard from the series all tubes falling outside the predetermined limits. At 
the high end of the galvanometer scale this should be not greater than plus or minus 0.2 
galvanometer units. For example, If Ihc readings cluster about 94. 7, the mode of the 
distribution Is 94.7; then, all tubes with readings less than 94.5 and greater than 94.9 
woulrl l« discarded. 

7. Pour out Ihc distilled water, dry as before and rinse with and fill 2/3 full of the 
permanganate solution. 

8. Keeping the same setting, record the transmissions of each tube as before. 
The rcadingH now will be between 30 and 40% transmlltancc. 

9. Again discard all tubes varying more than the preselected limits from the mode. 
For example if we decide to allow a variability of plus or minus 0. 1 galvanometer units 
and If the mode Is 35. C, then all lubes with readings less than 35.5 and greater than 
35.7 would be discarded. 

10. Let us calculate what this means In terms of inaccuracy contributed to the deter- 
mination by the phototubes . In order to calculate the optical densities we must correct 
for the fart thai Ihc blank lube was not act on 100 but on 94.7 (the mode of tiie distribu- 
tion). 94.7/35. G = 100/(37.6). (This is merely tlic calculation of the per cent trans- 
fiiHIance of the permanganate solution.) Now the optical density corresponding to a 

331 



transmit twice of 37.6 Is 0. <125 unci the tolerances allowed above renrettenl optical den- 
sity variations of 0.001 and 0.002 for the high and the low ends of the acale respective- 
ly. If these variations are additive they would represent a 0.7% error in the final 
optical density as calculated, If greater error can be tolerated, then wider tolerance 
limits can be set up. 

11. Precision bore tubing both cylindrical and square has recently become avail- 
able and this may help to solve this problem of calibration. All of this tubing, however, 
while of constant Internal thickness, has walls which vary In thickness and this Impairs 
their optical properties somewhat. 



VOLUME CALIBRATION OF TEST TUBES 



In many determinations It Is advantageous to dilute a solution to a given volume In 
a test tube. The test tubes may be calibrated by the following procedure: 

Arrange test tubes la a rack so that they can be easily handled. They need not be 
perfectly clean but must be dry. Using a volumetric transfer plpet, transfer quantita- 
tively the required volume of fluid such as 10 ml. ,16 ml. , etc. 

Arrange the tube In an apparatus, so that the tube can be held vertically and at the 
same time rotated about its long axis. 

Sighting across the lowest point of the meniscus (horizontally to avoid parallax) 
Bcratch opposite sides of the tube with a fixed diamond pencil. Recheck the accuracy 
of these points visually and then circumscribe the tube with the diamond point. Tubes 
may be numbered If required. 



332 






THE USE OF THE SLIDE RULE 



This section Is not Intended to substitute for the excellent manuals of Instruction 
which accompany most slide-rules. It Is Intended as a very brief statement regarding 
their use. Only persistent use of a slide rule will permit the owner to achieve that de- 
gree of familiarity which will serve to shorten the labor of mathematical calculations. 

Multiplication : 

The scale labeled C (on the slide) and the scale D (on the rule) are used for multi- 
plication. These two scaleB are exactly alike. The total length of these scales has been 
separated into smaller parts by graduations. Note that the distance between 1 and 2 and 
between 2 and 3 etc. , is not the same. It decreases as the number Increases. It 1b 
carefully measured to decrease logarithmically. So that what we do in multiplication Is 
to add lengths of the rule corresponding to the logarithm of the numbers we wish to 
multiply together. 

Remembering that the sum of the logarithm of A and B equals the logarithm of the 
product A x B 

log A / log B = log (A xB) 
We Bee that by adding lengths of rule we can multiply. 

Similarly, by subtraction of lengths of the rule, we can divide 
log A - log B - log (A/B) 
Example : Examples of simple multiplication and division will be given and your tech- 
nique can be checked at any time by redoing these examples. 

Multiplication : Multiply 2x3 
1. Setting the scales 

Set the left index (1) of the C scale on 2 of the D scale. Now find 3 on the C 
scale and read the product 6 on the D scale. Thus, the length for 2 plus the length for 
3 will be the length for the product (fl) . 

Division : Divide 8x4 

1. Find 8 on the D scale, Bet 4 on the C scale over It and read the result 2 on the 
D scale under the Index 1 of the C scale. The length for 8 minus the length for 4 is the 
length of the dividend (2). 

Decimal Point Location : 

The slide rule does not locate the decimal point. However in most cases the loca- 
tion of the decimal point can be estimated by "common" sense. Thus 122. 1 + 9. 6 is 
about 100/10 and the answer Is about 10 (12.73; not 1.273; not 127.3). 

Further details and short-cuts can be seen and learned by reference to slide rule 
manuals. 



333 



Common Lop,nriU\mn of Niimlmrs 






N _JL_ * __ 2 3 Jl_ 

10 oooo 00IJ3 00O6 0T2U 01 70 

11 OI4II4 Oh53 01(92 0531 0569 

12 0792 0828— O06U — 0099— 093li- 

13 1139 1173 1206 1239 1271 
11* 11*61 11192 1523 1553 150U 



15 1761 

16 20bl 
17 23014- 

18 2553 

19 2788 



1790 1816 161*7 1075 
2068 2095 2122 211*8 
-2330— 2355— 2380— 2lj05- 
2577 2601 2625 261(8 
2810 2833 2856 2678 



20 3010 3032 305lt 3075 3096 

21 3222 321*3 3263 3281* 3301* 

22 31,21, 3I4UU — 3U6U — 31*83 — 3502— 

23 3617 3636 3655 367U 3692 
21, 3802 3820 3838 3856 3871* 

25 3979 3997 liOllt 1*031 liOUB 

26 1*150 1(166 1*183 1*200 1*216 
27 1,3m 1*330— l*3l»6— 1*362—1*378- 

28 1*1*72 1*1*87 !*502 1*518 1*533 

29 l*62l* 1*639 1(651* U669 Ii683 

30 1*771 U786 1*800 1*811* U&29 

31 1*911* 14928 1*91*2 1*955 1*969 

32 5051 £065—5079—5092—5105- 

33 5185 5198 5211 5221* 5237 
31* 5315 5328 531*0 5353 5366 

35 51*1*1 51453 51*65 51*78 5190 

36 5563 5575 £587 5599 5611 
37 5682 569l*—5705— 5717—5729- 

38 5798 5809 5821 5832 581*3 

39 5911 5922 5933 59l*li 5955 

1*0 6021 60U 601*2 6053 6061* 
hi 6128 6138 611*9 6160 6170 

12 6232 621*3— 62"53— 6263-627!*- 

1*3 6335 631*5 6355 6365 6375 
1*1* 61*35 61*1*1* 6U514 61*61* 61*7!* 

1*5 6532 651*2 6551 6561 6571 
1*6 6628 6637 661*6 6656 6665 

1,7 6721 6730—6739—671*9—6756- 

ii8 6812 6821 6830 6839 681*8 
1*9 6902 6911 6920 6928 6937 

50 6990 6998 7007 7016 7021* 

51 7076 7081* 7093 7101 7110 

52 7160 7168—7177—7185—7193- 

53 721*3 7251 7259 7267 7275 
51* 732Ji 7332 73jiO 731(8 7356 

rf 12 3 "IT 

334 



5 6 7. _JL 9 

0212 02^J 0295 033II 037l* 
0607 061*5 0662 0719 0755 

-0969 lOOti— 1030--1072— 1106 

1303 1335 1367 1399 11)30 
1611* 161*1* 1673 1703 1732 

1903 1931 1959 1?07 2011* 
2175 2201 2227 2253 2279 

-21*30 21*55— 21*80— 2501*— 2529 

2672 2695 2718 27l*2 2765 
2900 2923 29l*£ 2967 2989 

3118 3139 3160 3181 3201 
3321* 33145 3365 3385 3I4OI4 

_3522 351,1—3560—357?-- 3598 

3711 3729 371*7 3766 3781* 
3892 3909 3927 391*5 3962 

1*065 I4O82 I4O99 I4II6 1*133 
1*232 1*21*9 1*265 1*281 1*298 

-1*393 1*1*09— kk 25—1*1*1*0—1*1*5^ 

1*5U8 a56l» 1*579 !*59l4 1*609 
b698 1*713 1*728 1*71*2 1*757 

1*81*3 1*857 UB71 14886 14900 
1*983 14997 5011 502^ 5038 

_£llo 5132— 51145— 5159— 5172 

5250 5263 5276 5289 5302 
5378 5391 51*03 51*16 51*28 

5502 5511* 5527 5539 5551 
5623 5635 56U7 5658 5670 

.57140 5752-5763—5775—5786 

5&55 5866 5877 5868 5899 
5966 5977 5988 5999 6010 

6075 6085 6096 6107 6H7 
6100 6191 6201 6212 6222 

-62814 6291*— 63014— 63ia— 6325 

6385 6395 61405 6hl5 61*25 
61*81* 61*93 6503 6513 6522 

6580 6590 6599 6609 6618 
6675 6681* 6693 6702 6712 

_6767 6776—6785—67914—6803 

6857 6866 6875 688x4 6893 
691*6 6955 6961* 6972 6901 

7033 701*2 7050 7059 7067 
7118 7126 7135 711*3 7152 

.7202 7210—7218—7226—7235 

7281* 7292 7300 7308 7316 
7361* 7372 7360 7388 7396 
~T~ 6 7 ~~S~ 9 



< 



Common Lo^ir lthmij oT Numbora 












H 





55 


76ol, 


% 


7662 


57- 


-7559 


58 


7636 


59 


7709 


60 


7782 


61 


7853 


62- 


— 7921* 


63 


7993 


66 


8062 


65 


8129 


66 


8195 


67- 


—8261 


68 


8325 


69 


8388 


70 


8151 


71 


8513 


72- 


.—8573 


73 


8633 


7b 


8692 


75 


8751 


76 


8808 


77- 


-8665— 


78 


8921 


79 


8976 


80 


9031 


81 


9085 


82- 


—9138 


83 


9191 


Bit 


9263 


85 


9296 


86 


93lt5 


87- 


—9395 


06 


961,5 


89 


fm 



_1 2 j__ Jl_ 

7612 7IU9 7627 7635 
7li90 7697 7505 7513 
7566— 7576— 7502-7589- 
761*2 761*9 7657 7661* 
7716 7723 7731 7738 

7789 7796 7803 7610 
7860 7866 7875 7802 
■7931 — 7938 — 79li 5 ■ -7952- 
8000 8007 8011* 8021 
8069 8075 8082 8089 

8136 811*2 811*9 8156 
8202 8209 8215 0222 
6267— 8276—8260—8287- 
8331 8338 831*1* 8351 
8395 61*01 61*07 81»lli 

81*57 81*63 81*70 81*76 
8519 8525 8531 8537 
8579—8585—8591" 8597- 
8639 8665 8651 8657 
8698 8701* 8710 8716 

8756 8762 8768 877li 
8811* 8820 8825 8831 
8871— 8676—8882- -068 7 - 
8927 8932 8938 8963 
6982 6987 8993 8998 



-5_ k- „1_ J_ _£__ 

71*1*3 7651 7659 71*66 7676 
7520 7528 7536 751*3 7551 

■7597 7601*- -7612- 7619— 7627 

7672 7679 7606 7696 77C1 
77U5 7752 7760 7767 7771* 

7818 7825 7832 7839 781*6 
7089 7896 7903 7910 7917 

-7959 7966—7973—7980—7987 

8028 8035 801*1 60l*8 8055 
8096 8102 8109 8116 8122 



8162 8169 8176 8182 8189 
8228 8235 821*1 62l*6 8256 

.8293 8299-8306—8312—8319 

6357 8363 8370 6376 8382 
81*20 01*26 8632 8639 8665 

8682 8688 8696 8500 8506 
8563 8569 8555 8561 8567 

-8603 8609— 8615—8621—8627 

8663 8669 8675 8681 8686 
8722 8727 8733 8739 8765 

6779 8785 8791 8797 8802 
8837 8862 8068 8856 8859 

-8093 8899—8906—8910—8915 

8969 6956 8960 8965 8971 
9006 9009 9015 9020 9025 

9063 9069 9076 9079 
9117 9122 9128 9133 
-9170-- 9175— 9180— 9186 
9222 9227 9232 9238 
9276 9279 9286 9289 

9325 9330 9335 9360 
9375 9380 9385 9390 
-9625- -9630—9635—9660 
9676 9679 9686 9689 
9523 9528 9533 9538 

90 9562 9567 9552 9557 9562 9566 9571 9576 9581 9586 

91 9590 9595 9600 9605 9609 9616 9619 9626 9628 9633 

92-— -9638 961*3—9667-9652-9657 9661 — >9666- -9671 -9675—9660 

93 9685 9689 9696 9699 9703 9708 9713 9717 9722 9727 
96 9731 9736 9761 9765 9750 9756 9759 9763 9768 9773 

9B 9777 9782 9786 9791 9795 
96 9823 9827 9832 9836 9861 

97 9868 9872—9877 9881- -9886 

98 9912 9917 9921 9926 9930 

22 2256 2261 2265 2262 mk 

M0 1 2 3 6 

335 



9036 


9062 


9067 


9053 


9058 


9090 


9096 


9101 


9106 


9112 


•9163- 


-9U49- 


-9156- 


-9159— 


-9165- 


9196 


?201 


9206 


9212 


9217 


9268 


9253 


9258 


9263 


9269 


9299 


9306 


9309 


9315 


9320 


9350 


9355 


9360 


9365 


9370 


■9600—9605- 


-9610-9615- 


—9620- 


9650 


9655 


9660 


9665 


9669 


9699 


9506 


9509 


9513 


9516 



9800 


9805 


9009 


9316 


9818 


9865 


9850 


9856 


9659 


9863 


9890- - 


■9096 


■9899- 


-9903- 


-9908 


9936 


9939 


996.3 


9966 


9952 


22Z& 


22fi3_ 


mi 


2221 


???6 


5 


6 


7 


8 


9 



Optical Dt-nslty (D) table for value b of per cent light trajiEmltled (T> 






0.0 0.1 



D = 2 - log 10 T, where T = JL x 100 



0.2 



0.3 



0.1 



0.6 



0.6 



0.7 



0.8 0.9 




I 
2 
3 
4 



2.000 
1.099 
1.523 
1.398 



3.000 
1.959 
1.678 
1.509 
1.387 



2.699 
1.921 
1.668 
1.495 
1.377 



2.623 
1.886 
1.638 
1.481 
1.367 



2.398 
1.854 
1.620 
1.469 
1.357 



2.301 
1.824 
1.585 
1.456 
1.347 



2.222 
1.796 
1.669 
1.444 
1.337 



2. 155 
1.770 
1.569 
1.432 
1.328 



2.097 
1.745 
1.553 
1.420 
1.319 



2.046 
1.721 
1.538 
1.409 
1.310 



1.301 1.292 1.284 1.276 1.268 1.260 1.252 1.244 1.237 1.229 



6 


1.222 


1.218 


1.208 


1.201 


1.194 


1.187 


1.180 


1.174 


1.167 


1.161 


7 


1.155 


1.149 


1.143 


1.137 


1.131 


1.125 


1.119 


1.114 


1.108 


1.102 


8 


1.197 


1.092 


1.086 


1.081 


1.078 


1.071 


1.066 


1.060 


1.056 


1.051 


b 


1.046 


1.041 


1.036 


1.032 


1.027 


1.022 


1.018 


1.013 


1.009 


1.004 



10 1.000 0.996 0.091 0.987 0.983 0.979 0.975 Q.971 0.967 0.963 



0.0 0.1 0.2 



0.3 



0.4 0.5 0.6 0.7 0.8 0.9 



( 



10 


1.000 


996 


991 


987 


983 


979 


976 


971 


967 


963 


11 


959 


955 


961 


947 


943 


939 


936 


932 


928 


924 


12 


021 


917 


914 


910 


907 


903 


900 


896 


893 


689 


13 


886 


883 


879 


876 


873 


870 


866 


863 


860 


857 


14 


854 


861 


848 


845 


842 


839 


836 


833 


830 


827 



15 



824 821 818 816 812 810 807 804 801 799 



16 


796 


793 


790 


788 


765 


783 


780 


777 


776 


772 


17 


770 


767 


764 


762 


769 


767 


764 


752 


750 


747 


18 


745 


742 


740 


738 


736 


733 


730 


728 


726 


724 


19 


721 


719 


717 


714 


712 


710 


708 


708 


703 


701 



20 



699 



697 695 693 690 638 



688 



684 682 680 



21 


678 


678 


674 


672 


670 


668 


668 


664 


662 


860 


22 


658 


666 


654 


652 


650 


648 


646 


644 


643 


640 


23 


638 


636 


635 


633 


631 


629 


627 


62S 


623 


622 


24 


620 


618 


616 


614 


613 


611 


609 


607 


606 


604 



336 



T 


0.0 


0.1 


0.2 


0.3 


0.4 


0.5 


0.6 


0.7 


0.8 


0.9 


25 


602 


600 


599 


597 


595 


593 


592 


590 


568 


587 


28 


585 


583 


582 


560 


578 


577 


575 


573 


572 


fi70 


27 


569 


567 


565 


564 


562 


561 


559 


558 


556 


1154 


28 


553 


551 


550 


548 


547 


545 


544 


542 


541 


539 


29 


638 


536 


535 


533 


532 


530 


529 


527 


526 


524 



30 



521 520 519 617 



516 



514 



513 



511 



510 



31 


509 


607 


506 


504 


503 


502 


500 


499 


498 


496 


32 


495 


493 


492 


491 


489 


488 


487 


485 


484 


483 


33 


481 


480 


479 


478 


476 


475 


474 


472 


471 


470 


34 


469 


467 


466 


465 


463 


462 


461 


460 


458 


457 


35 


450 


455 


453 


452 


451 


450 


449 


447 


446 


445 


36 


444 


442 


441 


440 


439 


438 


437 


435 


434 


433 


37 


432 


431 


429 


428 


427 


426 


425 


434 


423 


421 


38 


420 


419 


418 


417 


416 


415 


413 


412 


411 


410 


38 


409 


408 


407 


406 


405 


403 


402 


401 


400 


399 



40 



398 397 



390 



395 



394 



393 



391 



390 



389 



388 



41 


387 


380 


385 


384 


383 


382 


381 


380 


379 


378 


42 


377 


376 


376 


374 


373 


372 


371 


370 


369 


368 


4a 


307 


300 


305 


364 


363 


362 


361 


360 


359 


358 


44 


357 


350 


365 


354 


353 


352 


351 


350 


349 


348 



46 



347 340 345 344 



343 



342 341 



340 



339 338 



46 


337 


336 


335 


334 


333 


333 


332 


331 


330 


329 


47 


328 


327 


326 


325 


324 


323 


322 


321 


321 


320 


48 


319 


318 


317 


310 


315 


314 


313 


312 


312 


311 


49 


310 


309 


308 


307 


300 


306 


305 


304 


303 


302 



50 



301 300 299 298 298 297 



290 



295 



294 



293 



51 


292 


292 


291 


290 


289 


288 


287 


287 


286 


285 


52 


284 


283 


282 


281 


281 


280 


279 


278 


277 


277 


53 


270 


276 


274 


273 


272 


272 


271 


270 


269 


268 


54 


208 


207 


200 


205 


264 


264 


263 


262 


261 


260 



55 



260 269 



268 



257 250 256 255 



254 



253 253 



337 



0.0 



0.1 



0.2 



0.3 



0.4 



0.5 



0.6 0.7 



0.8 0.9 






56 


252 


251 


250 


249 


249 


248 


247 


246 


246 


246 


67 


244 


243 


243 


242 


241 


240 


240 


239 


238 


237 


58 


237 


236 


235 


234 


234 


233 


232 


231 


231 


230 


69 


229 


228 


228 


227 


226 


225 


225 


224 


223 


223 



60 



222 



221 220 



220 



219 218 



218 



217 



216 



215 



61 


215 


214 


213 


213 


212 


211 


210 


210 


209 


208 


62 


208 


207 


206 


206 


206 


204 


203 


203 


202 


201 


63 


201 


200 


199 


199 


198 


197 


197 


196 


195 


194 


64 


194 


193 


102 


192 


191 


190 


190 


189 


188 


188 



65 



187 186 166 186 



184 



164 



183 182 182 181 



66 


180 


180 


170 


178 


178 


177 


177 


176 


175 


176 


67 


174 


173 


173 


172 


171 


171 


170 


169 


168 


188 


68 


167 


167 


166 


168 


165 


164 


164 


163 


162 


162 


69 


161 


161 


160 


159 


159 


158 


157 


157 


166 


166 



70 



155 



154 



154 



153 



152 



152 



151 151 150 149 



71 


149 


148 


148 


147 


146 


146 


146 


144 


144 


143 


72 


143 


142 


141 


141 


140 


140 


139 


138 


138 


137 


73 


137 


136 


136 


135 


134 


134 


133 


133 


132 


131 


T4 


131 


130 


130 


129 


128 


128 


127 


127 


126 


126 



( 



75 



125 



124 124 



123 



123 



122 



121 



121 



120 120 



76 


119 


119 


118 


117 


117 


116 


116 


115 


116 


114 


77 


114 


113 


112 


112 


111 


111 


110 


110 


109 


108 


78 


108 


107 


107 


106 


106 


105 


106 


104 


103 


103 


79 


102 


102 


101 


101 


100 


100 


099 


099 


098 


097 



80 



097 096 



096 



09ft 



095 



094 094 093 093 



092 



81 


092 


091 


090 


090 


089 


089 


088 


086 


087 


087 


82 


086 


086 


086 


085 


084 


084 


083 


082 


062 


081 


83 


081 


080 


080 


079 


079 


078 


078 


077 


077 


076 


84 


076 


076 


076 


074 


074 


073 


073 


072 


072 


071 



86 



0T1 070 070 069 009 068 



068 



067 067 066 












338 






I 



T 

T 

85 


0.0 


0.1 


0.2 


0.3 


0.4 


0.5 


0.6 


0.7 


0.8 


0.9 


071 


070 


070 


069 


009 


068 


068 


067 


067 


066 


86 


060 


065 


064 


084 


063 


063 


062 


062 


061 


061 


8T 


060 


060 


069 


059 


058 


058 


057 


057 


057 


056 


88 


0S6 


055 


056 


054 


054 


Q53 


053 


052 


052 


051 


89 


051 


050 


050 


049 


049 


048 


048 


047 


047 


046 



90 



046 045 



045 



044 044 043 043 042 042 



041 



91 


041 


040 


040 


040 


039 


039 


038 


038 


037 


037 


92 


036 


036 


035 


035 


034 


034 


033 


033 


032 


032 


930 


032 


031 


031 


030 


030 


029 


029 


028 


028 


027 


94 


027 


026 


026 


025 


025 


025 


024 


024 


023 


023 



95 



022 022 021 



021 



020 020 



020 



019 019 



018 



m 


018 


017 


017 


016 


016 


015 


015 


015 


014 


014 


97 


013 


013 


012 


012 


Oil 


Oil 


Oil 


010 


010 


009 


98 


009 


008 


007 


007 


007 


007 




006 


005 


005 


99 


004 


004 


003 


003 


003 


002 




001 


001 


000 
































































339 



STANDARD ItKl'KKKHCK TEXTS 

Biggs fit Mat* Far lane, Human Blood Coagulation, Oxford, 1953, 1) lack well Scientific 

Publications. 
Gamble, James L. Kxtra -cellular Fluid, ed. 5, Cambridge, Ma.au. , 1052, Harvard 

University PresB. 
Gon/.alc-r., Vance, Hclpcm & Umberger, Legal Medicine and Toxicology, Ed. 2, 1954, 

Appleton-Century-Crofta, Inc. 
Hamilton, R. H. , and others. Plyslo logical CliemlHtry Laboratory Manual, ed. 7., 

Philadelphia, 1956, Temple University Sciiool of Medicine. 
Harper, Harold. A., Ph.D., Review of Physiological Chemistry, ed. 3, University 

Medical Publishers, P.O. Box 761, Palo Alto, California. 
Hawk, P.B., Osor, B.L. , and Summerson, W. H. , Practical Physiological Chem- 
istry, ed. 12, Philadelphia-Toronto, 1947, The Elakiston Company. 
Hoffman, W.S. , The Biochemistry of Clinical Medicine, Chicago, 1954, 

The Year Book PubUsliers. 
Knights, E.M., MacDonald, R. P. , and Ploompuu, J., Ultra -Micro Methods for 

Clinical Laboratories, Grune & Stratton, New York, 1957. 
Koch, Frederick C. , and Hanke, Martin E. , Practical Methods in Biochemistry, 

ed. 6, Baltimore, i953, The Williams and Wilkins Co. 
Kolmer, J. A. , Spaulding, E. H. , and Robinson, H. VV. , Approved Laboratory Technlc, 

ed. 5, New York, 1951, Appleton-Century Crofts, Inc. 
Levinson, S.A. , andMacFate, R.P. , Clinical Laboratory Diagnosis, ed. 5, Lea & 

Febiger, Philadelphia, 1956. 
Lippman, Richard W. , Urine and the Urinary Sediment, ed. 1, 1952, Springfield, El. 

Charles C. Thomas. 
Peters, J. P. , and Van Slyke, D. D. , Quantitative Clinical Chemistry, Vol II, Methods. 

Baltimore, 1932, Williams and Wilkins Co. 
Reiner, Miriam (ed.), Standard Methods of Clinical Chemistry, Vol I, (Amer. Assoc. 

Clin. Chemists), New York, 1953, Academic Press, Inc. 
Tocantins, Leandro M. , The Coagulation of Blood (Methods of Study), New York- 
London, 1955, Grune fir Stratton. 
Todd, J.C. , and Sanford. A.W. , Clinical Diagnosis with Laboratory Methods, ed. 10. 

PMladclplda and London, 1943, W. B. Saunders Co. 
Varley, Harold, Practical Clinical Biochemistry, ed. 1, New York-London, 1964, 

Interscience Publ. Inc. 
Wclsburg, Harry F. , Water, Electrolyte and Acid-Base Balance, Baltimore, 1953, 

Williams and Wilkins Co. 
Yearbook of Pathology and Clinical Pathology, Pathology Yearbook Publishers, Inc. 

Chicago, HI. 



340 






lURUOGRAPHY 



Aln.Hwoitlt. A. W. ; Analytical and Hi if rebalances, Ind. and Kny t . Cliom. (Anal. 
Ed.), J2. 572-3 (1939). 

Albanese. A, A. and V. lrby; Determination of urinary amino nitrogen by the 
copper method, J. Biol. Chum., 153 . 583-8 (1944). 

Albanesc, A. A. and V. Irby; Determination of amino nitrogen of blood filtrates 
by the copper method, J. Lab. and Clin. Med, , 30, 718-21 (1945). 

Algcri, K.J. andJ.T. Walker; Paper chromatography for Identification of the 
common barbiturates, Am. J. Clin. Path., 22, 37-40 (1952). 

Alper, C. , Lipase- (Tributyrlnase). In: Standard Methods of Clinical Chemistry 
(Vol. 1), Academic Press Inc., Publishers, New York, 1953, pp. 71-4. 

Altman, V. , M. Nelson and D. Pernova, Determination of plasma bilirubin by a 
simple mlcromethod. Am. J. Clin. Path., 26, 956-2 (1956). 

Anderson, G.E. , It. W. Hlllman, I.F.A. Van Elk and A. J. Perfltto; Postabsorp- 
tlve undulations and oscillations In blood glucose. Am. J. Clin. Nutr. , 4, 673-86 
(1956). 

Anker, R.M. : Determination of estrogens In stored urines of pregnancy, J. of 
Clin. Endocr. and Metabolism, 15, 210-14 (1955). 

Archibald, R.M.; Determination of lipase activity, J. Biol. Chem. , 165 , 443-8 
(1946). 

Aakevold, R. ; Routine analysis of porphyrins in urine, Scand. J. of Clin, Invest. , 
3, 318-9 (1951). 

Bachman, C. and D.S. Pettit; Photometric determination, of estrogens, J. Biol. 
Chem., 138, 689-704 (1941) , 

Bamford, F. , C. P. Stewart and S. Smith; Poisons, Their Isolation and Identifica- 
tion (3rded.), The Blakiston Co. , Philadelphia, 1951, 11 pp. 68-71. 

Barker, S.B., M.J. Humphrey and M.H. Holey; The clinical determination of pro- 
tein bound iodine, J. Clin. Invest. , 30, 55-62 (1951). 

Barnes, R.B., D. Richardson, J.W. Berry and R. L. Hood; Flame photometry, 
Ind and Eng. Chem. (Anal. Ed.), 17, 605-11 (1945). 

Bergmann, F. , and S. Dikstein; Studies on uric acid and related compounds, J. Biol. 
Chem. , 211, 149-53 (1954). 

Berry, J.W. , D. G. Chappcli and R.B. Barnes; Improved method of flame photo- 
metry, Ind. and Eng. Chem. (Anal. Ed.), Jji, 19-24 (1946). 

Blade, E. ; Calibration of weights, Ind. and Eng. Chem. (Anal. Ed.), 11, 499-501 
(1939). 

Bloor, W.R. and A. Knudson; The separate determination of cholesterol and chol- 
esterol esters In small amounts of blood, J. Biol. Chem., 27, 107-12 (1916). 

Bloor, W.R., K. F. Pclkan and D. M. Allen; Determination of fatty acids (and 
cholcHtcrol) In smalt amounts of blood plasma, J. Biol. Chem, , 52, 191-205 
(1922). 



Bogon, K.: A quantitative study of acute alcoholic intoxication, Am. J, Med. Scl., 
176, 153 (1928). 

Boutwntl, J. H. , Jr. ; The determination of urea as ammonia by direct nesslcrlza- 
Hon, Clin. Clietn., 3, 205 (1957). 

Bowler, H.G. The determination of thlocyanate In blood serum, Blochom. J., 38, 
385-8 (1041). 

Bratton, A. C. , andE.K. Marshall, Jr.; A new coupling component for sulfanil- 
amide determination, J. Biol. Chem. , 128, 537-50 (1939). 

Briggs , A. P. A colorlmctric method for determination of small amounts of 
magnesium, J. Biol. Chcm. , 52, 340-355 (1922). 

Brodle, B.B., S. Undcnfricnd and H. F. Coburn; Determination of salicylic acid 
in plasma, J. Pharmacol, and Exp. Therapeutics, 80. 114-7 (1944), 

Brown, H.; The determination of uric acid in human blood, J. Biol. Chem. , 158 , 
601-8 (1945). 

Brunsting, L. A., H. L. Mason and R. A. Aldrich; Adult form of chronic porphyria 
with cutaneous manifestations, J. A.M. A., 146, 1207-12 (1951). 

Buchanan, O.H. , W.D. Block and A. A. Christman; The metabolism of the methy- 
lated purines , J. Biol. Chem., 157 , 181-7 (1945). 

Bunch, L.D. , and R. L. Emerson, Serum lipase determination; four hour technlc 
with olive oil substrate, Clin. Chem, , 2, 75-82 (1956), 

Cabaud, P. , R. Leeper and F. Wroblewski; Colorimetric measurement of serum 
glutamic oxalacetic transaminase, Am. J. Clin. Path. , 26, 1101-6 (1956). 

Callow, N. , R. Callow and C. Emmens; Colorimetric determination of substances 
containing the grouping -CH 2 CO~ in urine extracts as an indication of androgen 
content, Biochem. J., 32, 1312-31 (1938). 

Carr, J.J. and I.J. Drekter; Simplified rapid technlc for the extraction and deter- 
mination of serum cholesterol without saponification, Clin. Chem. , 2, 353-68 
(1956). 

Cherry, I.S., and L, A, Crandall; The specificity of pancreatic lipase: its appear- 
ance in the blood after pancreatic injury, Am. J. Physiol. , 100, 266-73 (1932). 

Clark, W.M., The Determination of Hydrogen Ions (3rd ed.): Williams and Wilkins, 
Baltimore, Md. , 1928. 

Clark, E. P. and J.B. Collip; A study of the Tlsdall method for the determination of 
blood serum calcium witha suggested modification, J. Biol. Chem. , 63, 461-4 
(1925). 

Clark, L. C. , Jr. and H. L. Thompson; Determination of creatine and creatinine in 
urine, Anal. Chem., 21, 1218-21 (1949). 

Connerty, H.V.\ A.R. Brlggs and E. H. Eaton; Determination of blood urea nitro- 
gen using a simple stabilizing reagent, Am. J. Clin. Path., 25, 1321-5 (1955). 

Craig, A.; The testing of chemical balances'? Ind, and Eng. Chem. (Anal. Ed.), 11, 
581-2 (1939) 

Crooke, A.C., W.R. Butt, J. D. Ingram and L. E. Romanchuck; Chemical assay of 
gonadotropins in urine, Lancet, I, 379-83 (1954). 

Cutler, H. H. , M.H. Power and R.M. Wilder; Concentrations of chloride, sodium 
and potassium In urine and blood, J. A.M. A. , 111, 117-22 (1938). 

Darling, R.C. Electrolyte abnormalities of the sweat In fibrocystic disease of the 
pancreas, Am. J. Med. Sci. , 225, 67 (1953). 

342 



Davhtsohn, 1. oil.) , A curriculum for Schools of Medical Tcolinoiof.y (;ird ml.): 

Registry of Medical Technologists, American Society or Clinical Pathologists, 

Muncie, Iud. , 1953. 
Davis, U.J. and E. Wood; Improved method for prostatic acid phosphatase, Clin. 

Chem. , 2. 2-18 (1956). 
Dckanskt, J, ; The kaolin adsorption method for the quantitative assay of urinary 

gonadotropins, Brit. J. Exp. Path., 30, 272-82 (194D). 
Denis, W. The determination of magnesium in blood plasma and scrum, J. Biol. 

Chem. , 52, 411 (1922). 
Diethelm, O. , On bromide Intoxication, J. Ncrv. and Mental Dis. , 71, 151-65 

(1930). 
di Sant'Agncse, P. A. ; Abnormal electrolyte composition of sweat in cystic fibrosis 

of the pancreas : its clinical significance and relationship to the disease. 

Pediatrics, 12, 549 (1953). 
Dragstedt, C.A. and M. A. Mills; The employment of oxaiated plasma in the brom- 

sulphaleln dye retention test, J. Lab. and Clin. Med, , 21, 1306-7 (1936). 
Dryer, R.L. , A.R. Tammes and J.I. Routh; The determination of phosphorus 

and phosphatase with N-phenyl-p-phenylenedlamlne, J. Biol. Chem., 225 , 

177-83 (1957). 
Ducci, H. ; The thymol test of Mac lagan, J. Lab. and QUn. Med., 32, 1266-72, 

(1947). 
Duccl, H. and C.J. Watson; The quantitative determination of the serum bilirubin 

with special reference to the prompt-reacting and the chloroform soluble types, 

J. Lab. and Clin. Med., 30, 293-300 (1945). 
Dumm, R.M. andR.A, Shipley; The simple determination of blood ketones In 

diabetic acidosis, J. Lab. and Clin. Med. jl, 1192-3 (1946). 
Engstrom, W. and H. Mason; A study of the colorimetric assay of urinary 17- 

ketosterolds, Endocrln. , 38, 229-38 (1953). 
Ferro, P.V. andA.B. Ham; The spectropliotometric determination of calcium, 

In press. 
Finlay, J.M. , B.E.C. Nordln and R. FraBer; A calcium Infusion test, Lancet, 

1, 826-30 (1956). 
Flshman, W. H. and F. Lerner; A method for estimating serum acid phosphatase 

of prostatic origin, J. Biol. Chem., 200, 89-97 (1953). 
Flske, C. H. and Y. Subarrow; Colorimetric determination of phosphorus, J. Biol. 

Chem., 66^ 375-400 (1925). 
Flster, H.J. , Manual of Standardized Procedures for Spectrophotometry Chem- 
istry: Standard Scientific Supply Co., New York, 1950. 
Folin, O. and H. Wu; A system of blood analysis, J. Biol. Chem. , 38, 81-110 

(1919). 
Fowell, A.H. ; Turbidimetrlc method of fibrinogen assay , Am. J. Clin. Path,, 

25, 340-2 (1955). 
Fowweather, F.S. and W.N. Anderson; A method for the determination offat in 

feces, Blochem. J. , 40, 350-1 (1946). 



343 



Franco, V. and 11. Klein: The ntii.' redetermination of chlorides In scrum and 

spinal fluid, J. Lab. and Clin. Med. , 37, 950-4 (1951). 
Gai'blor, O. II. : Determination of bromsulphalcin in normal, turbid, hcmolyzed or 

Icteric scrums, Am. J. CHn. Path., 15, 452-5 (1945). 
Glolster, J. , Medical Jurisprudence and Toxicology (9th ed.): E. and S. Living- 
stone, Ltd.. 1950, pp. 545-7. 
Goldbaum, L.R. Determination of barbiturates, Anal. Chem. , 24, 1604-7(1952). 
Goldstein, N. P. , J. P. Epstein and J. H. Roe; Studies of pancreatic function, 

J. Lab. and Clin. Med., 3jL 1047-51 (1948). 
Gomorf, G. ; A modification of the colorimetric phosphorus determination for use 

with the photoelectric colorimeter, J. Lab. and Clin. Med., 27, 955-60 (1942). 
Gomorl, G. ; Assay of pancreatic lipase in Berum, Am. J. Clin. Path. , 27, 

170-82 (1957). 
Gomorl, G. ; The determination of amylase In small amounts of serum, Am. J. 

Clin. Path.,_27, 714 (1957). 
Goodman, R.D. andG.R. Klngsley; Sulfobromophthaleln clearance test, J.A.M.A. 

153 , 462-6 (1953). 
Goodman, L. and A. Gilman, The Pharmacological Basis of Therapeutics: 

McMillan Co, , New York, 1941, p. 23. 
Gorden, H. and N,L. Larson; Use of sequestrene aB an anticoagulant, 

Am. J. Clin. Path., 23, 613-8 (1953). 
Gornall, A,G. , C.J. Bardawlll and M. M. David; Determination of serum proteins 

by means of the biuret reaction, J. Biol. Chem. , 177 , 761-fld (1949). 
Gradwohl, R.B.H., Legal Medicine : C.V. Mosby Co. , St. Louis, 1954, p. 598. 
Gradwohl, R.B.H. , Chemical Laboratory Methods and Diagnosis (5th ed.): 

C.V. Mesh Co., St. Louis, 1956, pp. 551-3. 
Greenberg, L. A. and D. Lester; A micromethod for the determination of acetone 

and ketone bodies, J. Biol. Chem., 154, 177-SO (1944). 
Haden, R. L. ; A modification of the Folln-Wu method for making protein-free blood 

filtrates, J. Biol. Chem. , 56, 469-71 (1923). 
Hadley, G.G. and S. P. Weiss; Further notes on use of salts of ethylene -diamine 
tetraacetic acid (EDTA) as anticoagulants, Am. J. Clin. Path. , 25, 1090-3 
(1955). 
Hall, W. W. ; Drunkenness, naval medico-legal aspects of the diagnosis, U.S. Navy 

Medical Bull. , 34, 149-63 (1936). 
Hamilton, R.H. ; Photometric determination of bromsulphaleln In hcmolyzed, 

lipemlc or icteric serum, Fed. Proc. , 6, 258 (1947). 
Handbook of the Hospital Corps, U.S. Navy: U. S. Government Printing Office, 

Washington, D.C., 1953. 
Hanger, F.M. ; Serological differentiation of obstructive from hepatogenous 

jaundice by flocculation of cephalln -cholesterol emulsions, J. Clin. Invest. , 
18, 261-9 (1939). 
Hanger, F.M. and A. J. Patek, Jr.; The cephalln flocculation teat in cirrhosis of 
the liver, Am. J. Med. Sci. 202, 48-52 (1941), 



344 



( 



Heagy, F.C. . Uh<- or polyvinyl alcohol In the colorl metric determination of magne- 

sium In plasma or scrum by nicami of til;ui yellow. Can. J. Research, 26E 

295 (11)48). 
Henry, H. J. , C. Sobel and S. Berkmann; On the determination of "pancreatic 

lipa.se" In serum, Clin. Chem. , 3, 77-89 (1957). 
Henry. H.J., C. Sobel and M. Seaglove^ Turbidlmetrlc determination of proteins 

wltli sulfosalicytie and trichloracetic acids, Proc. Soc. Exp. Biol, and Med. , 

92, 748-51 (1956). 
Hepler, O. E. , Manual of Clinical Laboratory Methods (4lh ed.) : Charles C. 

Thomas, Springfield, 111. , 1949. 
Hiller, A. , J. Plaxin and D.D. Van Slyke; A study of conditions for Kjcldahl 

determination of nitrogen in proteins, 176 , 1401-20 (1048). 
Hoffman, W, S. and B. Osgood; The photoelectric mic rode termination of nitrogen- 
ous constituents of blood and urine by direct Ncsslerlzatlon, J. Lab. and 

Clin. Med., 25, 856-62 (1940). 
Huerga, J. De la and H. Popper; Standardised reagent for thymol turbidity test, 

J. Lab. and Clin. Med. , 34, 877-80 (1940). 
Huggins, C. and P.S. Russell; The determination of amylase, Ann. Surg,, 128 , 

668 (1948). 
Hulett, G.A. and W. D. Bonner, A method for preparing standard hydrochloric 

acid solutions k J. Amer. Chem. Soc, 31, 390-3(1909). 
Hunter, G. ; On the bromide test of permeability of the barrier between blood and 

cerebrospinal fluid - an assessment, Biochem. J. , 56, C88-97 (1954). 
Hurley, F. H. , Jr. ; Borax as an acldimetrlc standard, Ind. and Eng. Chem. 

(And. Ed.),_8, 220-221 (1936). 
Hurley, F.H., Jr.; Borax as an acidimetric standard. II, Ind. and Eng. Chem. 

(Anal. Ed.), 9, 237-238 (1937). 
Karr, W. G. ; A method for the determination of blood urea nitrogen, 

J. Lab. and Clin. Med. , 9, 329-33 (1924). 
Katzenelbogcn, S. and T. Szarski; Improved colorimetrlc method for determination 

of bromide concentration in blood and cerebrospinal fluid, Pro, Soc. Exp. Biol, 

and Med. , 32. 136-7 (1934). 
Kaye, S. , Handbook of Emergency Toxicology: Charles C. Thomas, Springfield, 

M. , 1954, pp. 134-40. 
Kind, P. R. N. , and E. J. King; Estimation of plasma phosphatase by determination 

at hydrolyzed phenol with amtno-antipyrine, J. Clla. Path. , 7, 322-6 (1954). 
King, E. J. and D. S. Bain; A simplified silver iodate method for the determination 

of cMoride, Biochem. J., 48, 51-3(1951). 
Klugerman„ M.R. ; A simple and rapid calculation In Barker's method for blood 

prote to-bound iodine, Am. J. Clin. Patfo. r j!4 r 490-5 (1954), 
Kunkel, H, G. , Estimation of alterations of serum gamma globulin by a turbidimetric 

technique, Proc. Soc. Exp. BloL and Med. , 66, 217-24 (1047). 
Levlnson, S.A. and R.P. MacFate, Clinical Laboratory Diagnosis (lsted.): 

Lea and Febigcr, Philadelphia, 1937, pp. 176-81. 



345 






Levy, M.S., M.H. Power and E. J. Kepler; Ttic specificity ol the "water-test" 

as 11 diagnostic procedure in Addison's disease, J. Clin. Endocr, and 

Metabolism, G, G07-32 (194G). 
Lon E , II. P., Modem Med., Nov. 1 (1954). 
Looney, J.M. and A.I. Walsh; The determination of spinal fluid protein with the 

photoelectric colorimeter, J. Biol. Chem. , 127, 117-21 (1939). 
Lorlion, F. ; On the determination of creatinine tn plasma by the Jaffe reaction, 

after adsorption to tlie Lloyds reagent, Scand, J. Clin, invest. , 6, 325.-34 (1955). 
de Lourclro, J. A. and G.J. JaiiK; lodometrlc and colorimetrlc methods for the 

estimation of calcium tn serum based on (he use of an Improved permanganate 

solution, niochem. J., 38, 16-9 (1944). 
Malloy, H.T. andK.A. Evelyn; The determination of bilirubin with the photoelectric 

Colorimeter, 3. Biol. Chem., Hi. 481-90(1937). 
Mandel, E.E., E.B. Lehmann and D. A. Paris; U.S. Public Health Service, Com- 
municable Disease Center, Federal Security Agency, March, 1950. 
Maren, T. H. ; A simple and accurate method for the determination of mercury in 

biologic material, J, Lab. and Clin. Med. , 28, 1511-4(1943). 
McNally, W.D. , Toxicology: Industrial Medicine, Chicago, 1837, p. 1. 
Mitzner, B.M. ; Drying volumetric glassware, J. Chem. Ed. , 33, 609 (1958). 
Nelll, D. W. and R.A. Ncoly; The estimation of magnesium In serum using 

titan yellow, J. Clin. Path. , 9, 162 (1956). 
Nelson, N. ; A photometric adaptation of the Somogyi method for the determination 

of glucose, J. Biol. Chem., 153, 375-80 (1944). 
Newman, H. W. and N.J. Aahcnburg; Chemical diagnosis of drunkenness, 

U. S. Navy Med. Bull. , 44, 744-5 (1945). 
Official Methods of Analysis of the Association of Official Agricultural Chemists 

(7th ed.), Associationof Official Agricultural Chemists, Washington, D.C., 

1950, p. 369. 
Owren, P. A.; Prothrombin and accessory factors, Am. J. Med., W, 201 (1053). 
Ozburn, E. E. ; A rapid method for determining methyl alcohol in the blood and 

body fluids, U.S. Navy Med. Bull., 46, 1170-3 (1946). 
Parfentjev, I. A. , et al; Determination of plasma fibrinogen by turbidity with 

ammonium sulfate, Arch. Bloc hem, , 46, 470-80 (1953). 
Patton, J. and W. Reeder; Indicator for titration of calcium with (othylenedlnitrilo)- 

tetraacetate, Anal. Chem., 28, 1026-8 (1956). 
Paumz, L. ; Diagnosis of amyloidosis by means of Congo red, Magyar Orvosi 

Archlvum, 25, 499-502 (1924): Chem. Aba. , 19, 1009 (1925), 
Peralta, O. andJ.G. Reinhoid; Rapid estimation of amylase activity of serum 

by turbldimetry, Clin. Chem. , 1, 157-64 (1955). 
Peters, J. P. and D.D. Van Slyke, Quantitative Clinical Chemistry (2nded.): 

Williams and Wilkins, Baltimore, 1946, Vol. 1, Chapt. 18. 
Powell, M. E. A. and M.J. H. Smith; The determination of serum acid and alkaline 

phosphatase activity with 4-amlnoantipyrine (AAP), J. Clin. Path., ,7, 245-8 

(1954). 



346 



Quick. A. J.; The determination of prothrombin activity, Am. J. Clin. Path,, 

jjO, 222 (1U40). 
Rehell, U.; A rapid clinical method for the determination of calcium in serum and 

other biological fluids, Scand. J. Clin, Invest,, 6, 335-40 (1955). 
Richards, T.W. j Note on a method of standardizing weights, J, Am. Chem. Soc. , 

22, 144-9 (1900). 
Robinson, F.J., M.H. Power and E, J. Kepler; Two new procedures to assist in 

the recognition and exclusion of Addition's disease: a preliminary repor', 

P l0 c. Staff Meeting of the Mayo Clinic, J^, 577-83 (1941). 
Roe, J.H. and CA. Kuether; The determination of ascorbic acid In whole blood 

and urine through the 2,4-dinltrophenylhydrazine derivative of dehydroascorbic 

acid, J. Biol. Chem., 147, 399-407 (1943). 
Rosenberg, A. A. ; Plasma fibrinogen determination; a rapid titration method, 

Clin. Chem,, 2_, 331-3(1956). 
Rosenthal, H.L, and H.I. Cundiif; New biuret reagent for the determination of 

proteins In cerebrospinal fluid, Clin. Chem., 2, 394-400 (1956). 
Rosenthal, R.L., O. H. Dreskln and N. Rosenthal; Plasma thromboplastin ante- 
cedent (PTA) deficiency: clinical, coagulation, therapeutic and hereditary 

aspects of new hemophilia-like disease, Blood 10, 120 (1955). 
RoBenthal, S.M. and B.C. White; Clinical application of the bromsulphalein test 

for hepatic function, J. A.M. A., 84. 1112-4 (1825). 
Roughton, F.J.W. and P.F. Scholander; Mlcro-gasometric estimation of the blood 

gases, J. Biol. Chem. , 149, 651-63 (1943). 
fialfer, A. and J. Hughes; Determination of chlorides in biological fluids by the 

uee of adsorption indicators, J. Biol. Chem. , ;12S, 273-81 (1939). 
Sandell, E. B. ; Colorlmetric mlc redetermination ojf arsenic after evolution as 

arulne, Ind. and Eng. Chem. (Anal, Ed.), 14, 8a-3 (1942). 
BandeH, E.B., Colorlmetric Determination of Traces of Metals: (2nded.): 

lnterscience Publishers, Inc., New York, 1950. 
Schales, O. andS.S. Scliales; A simple and accurate method for the determination 

of chloride In biological fluids, J. Biol. Chem., 140 , 879-84 (1941). 
Schmidt, C. H. , M.E. Hane and D.C. Gomez; A new anticoagulant for routine 

laboratory procedures, U.S.A.F. Med. J., 4, 1556-62 (1953). 
Bchoenhelmer, R. and W. M. Sperry; A micromethod for the determination of free 

and combined cholesterol, J. Biol. Chem., 106, 745-60 (1934). 
Schaffert, R.R, andG.R. Kingsley;A rapid, simple method for the determination 

of reduced, dehydro-, and total ascorbic acid in biological material, J. Biol. 

Chem., 212, 59-68 (1965). 
Schroeder, W.A. , L.M. Kay andR.S. Mills; Quantitative determination of amino 

aclda by iodometric titrations of their copper salts, Anal. Chem. , 22, 760-3 

(1950). 
Shank, R. E. and C.L. Hoagland; A modified method for the quantitative determina- 
tion of the thymol turbidity reaction of serum, J. Biol. Chem. , 162, 133-8 

(1946). 



347 



Shilling, A. and D. L.i/lo, Kale of urinary calcium excretion following Intravenous 

Infusion, Proc. Sue. Kxp. Biol. L Med., 78, 286 (1951). 
Shipley, it, A. and C. N. II. Long; Studios on the ketogcnlc activity of the anterior 

pituitary, Blochcm. J. . 32, 2212 (1938). 
Sllverstone, F T A. , W. Brandfonbrener, N.W. Shock and M.J. Ylcngat; Age differ- 

enceB In the Intravenous glucoee tolerance tests and the response to insulin, 

J. Clin. Invest. , 3G, 504-14 (1957). 
SJoerdama, A., 11. Wclssbach and S. Udenfrlend; Simple teBt for diagnosis of 

metastatic carcinoid (arg^ntafrinoma) , J.A.M.A., 159, 397 (1955). 
Smith, B. W. and J. It. Roe; A photometric method for the determination of a- 

amylase In blood and urine with use of the starch iodine color, J. Biol. Chem. , 

179 , 53-9 (1949). 
Smith, B.W. and J.H. Roe; The micro-determination of amylase, J. Biol. Chem., 

227 , 357 (1957). 
Smith, L. E. and N.W, Shock; Intravenous glucose tolerance tests in aged males, 

J. Gerontology, 4, 27-33 (1949). 
Somogyl, M. , Mlcromethods for the estimation of diastase, J. Biol. Chem., 125, 

399-414 (1938). 
Somogyl, M. ; A new reagent for the determination of sugars, J. Biol. Chem. , 

160 , 61-8 (1945). 
Somogyl, M. ; Determination of blood sugars, J. Biol. Chem., 160, 69-73(1945). 
Sperry, W. M. and M. Webb; A revfsion of the Schoenheimer-Sperry method for 

cholesterol determination, J. Biol. Chem. , 187, 97-110 (1950). 
Steinberg, D. , D. Baldwin and B. H. Ostrow; A clinical method for the assay of 

serum glutamic oxalacctlc transaminase, J. Lab. and Clin. Med., 46,144-61 

(1956). 
Sussman, L.N. , LB. Cohen and R. Gittler; Serum prothrombin (prothrombin 

consumption test), J. A.M. A., 156, 673-75 (1954). 
Shwachman, H, , E. Higglns and R. R. Dooley; A simple diagnostic test for muco- 
viscidosis: Sweat electrolyte studies, In preparation. Preliminary observa- 
tions presented before the American Academy of Pediatrics," October 6. 1954, 

Chicago, 111. 
Shwachman, H. and H. Leubner; Mucoviscidosis, Advances in Pediatrics, Year- 
book Publishers, Chicago, Vol. VII, 1955. pp. 249-323. 
Taussky, H. H. ; A mlcrocolorimetric determination of creatine in urine by the 

Jaffe reaction, J. Biol. Chem., 208 , 853-61 (1954). 
Taussky, H. H. ; A procedure Increasing the specificity of the Jaffe reaction for the 

determination of creatine and creatinine In urine and plasma, Cllnlca Chlmlca 

Acta, 1, 210-24 (1956). 
Thienes, C. H. and T.J. Haley, Clinical Toxicology (2nd ed.): Lea and Feblger, 

Philadelphia, 1948. 
Thompson, H. L. , M.R. Klugerman and J. Truemper; A method for protein -bound 

iodine: the kinetics and use of controls in the ashing technique, J. Lab. and 
Clin. Med. . 47, 149-63 (1956). 



348 



Udcnfricnd, S. , 11. Wcistibach and C. Clark; The estimation of 5-hydroxylrypla- 

mine (serotomin) in biological tissues, J. Biol. Chem. , 215 , 3117-44 (1955). 
Udcnfricnd, S., II. Weissbach and E. Titus; The Identification of 5-hydroxy-3- 

lndolacellc acid in normal urine and a method for iln assay , J. Biol. Chem. , 

216, 499-506 (1955). 
Van de Kamer, J. H. , H.T. Huinlnkand H.A. Weyers; Rapid method for the 

determination of fat in feces, J. Biol. Chem. , 177, 347-55 (1949). 
Van Ptlsum and M. Bovis; Effect of various protein preclpltants on recoveries of 

creatinine added to plasma, Clin. Chem. , 3, 90-4 (1957). 
Van Slyke, D.D. , A method for the determination of carbon dioxide and carbonates 

in solution, J. Biol. Chem. , 30, 347-68 (1917). 
Van Slyke, D.D. and G.E. Cullen; The bicarbonate concentration of the blood 

plasma; its significance, and its determination as a measure of acidosis, 

J. Biol. Chem., 30, 289-346 (1917). 
Van Slyke, D.D. and J. A. Hawkins; A gasometric method for determination of 

reducing sugars, and its application to analysis of blood and urine, J. Biol. 

Chem., 79, 739-67 (1928). 
Varley, H. , Practical Clinical Biochemistry; Intersclence Publishers, Inc. , 

New York, 1954, p. 324. 
Ventura, S. ; Determination of the unsaturated iron -binding capacity of serum, 

J. Clin. Path. , 5, 270-4 (1952). 
Watson, C.J. and S. Schwartz; A simple test for urinary porphobilinogen, 

Proc. Soc. Exp. Biol, and Med. , 47, 393-4 (1941). 
Webster, W. W. , Legal Medicine and Toxicology: W. B. Saunders Co. , Philadelphia, 

1930, p. 318. 
West, E.S. andW.R. Todd, Textbook of Biochemistry (2nd ed.): MacMIUan Co. , 

New York, 1955, pp. 485-7. 
Whitehorn, J.C.; A system of blood analysis, J. Biol. Chem., 45, 449-60 (1921). 
Wilson, D. W. and E.G. Ball; A study of the estimation of chloride in blood and 

serum, J. Biol. Chem., 79, 221-7 (1928). 
Winer, J. H. and M.R. Mattice; Routine analysis of urinary calculi. A simple 

rapid method using spot tests, J. Lab. and CUn. Med. , 28, 898-904 (1942). 
Wroblewski, F. and P. Cabaud; Colorlmetric measurement of serum glutamic 

pyruvic transaminase. Am. J. Clin. Path., 27, 235-9 (1957). 
Wroblewski, F. and J.S. La Due; Serum glutamic pyruvic transaminase in cardiac 

and hepatic disease, Proc. Soc. Exp. Biol, and Med. , 91, 569-72 (1956). 
Zleve, L. , E. Hill, M. Hanson, A.B. Falcone and C.J. Watson; Normal and 

abnormal variations and clinical significance of the one -minute and total serum 

bilirubin determinations, J. Lab. and Clin. Med. , '38, 446-69(1951). 



349 



INDfcX 



( 









Absolute temperature 
Accelerator globulin 

accelerln 
Aceto-acetic acid 

effect on acid-base balance 
Acetone 

Acetylcholinesterase 
Actds, bases and buffers 

definitions 

pH, calculation 
definition 
scale 

primary standards 

water, ionization of 
Acid-Base equilibrium 

alterations in 
Acidosis, CG 2 in 
Acromegaly, glucose tolerance 
Acute abdomen, acetone in 
Acute Infection, fibrinogen in 
Addison's disease 

acid-base equilibrium in 

glucose tolerance In 

Kepler-Power water teBt in 

17-keto-steroids in 
Adrenal cortical malfunction 

glucose tolerance in 

17-keto-steroids fn 
Adrenal insufficiency test 
Albumin, and A/G ratio 

effect on thymol turbidity 
Albuminuria 
Alcohol, ethyl 

intoxication 
Alcohol, methyl 
Alcoholism, chronic 

magnesium in 
Aliesterase 
Afkall reserve 
Alkalosis 

CO z in 



63 


A u u u o acid i 


93 


244 


Ammonia, nine, method 


267 


324 


in acid-tsse equilibrium 


321 




Amylase 


97.101 


322 


Amyloidosis 




83 


Congo red teBt In 


168 


165 


Analysis 




39 


general methods of 


3 


3d 


control 


316 


42 


statistical 


317 


40 


Analytical balance 


6 


40 


Anemia 


304 


20 


serum iron in 


197 


39 


iron-binding capacity In 


200 


129,321 


Anions, non-volatile 




322 


in acid-base equilibrium 


322 


130 


Anterior pituitary disease 




184 


glucose tolerance In 


184 


84 


17-keto-steroids in 


206 


178 


Anti -coagulants 






circulating 


328 


322 


laboratory use 


73 


184 


therapeutic 


328 


85 






206 


Anti -fibriftoly Bin 


326 




Anti -hemophilic globulin 


324 


184 


Anti -thrombin 


326 


206 


Anti -thromboplastin 


326 


85 


Apparatus 




237 


colorlmetrlc 


48 


257 


gasometric 


132a 


304 


photometric 


58 


88 


volumetric 


11 


90 


Argentimetric primary standard 


23 


91 


chloride method's 


147,148 




Arsenic 


279 


210 


Ascites 


304 


165 


Ascorbic acid 


103 


130 


saturation test 
Aub diet 


106 


130 


for calcium balance studies 


125 



( 



350 



c 



I'aktncei:, lypctt, operation 

wvlglitii, calibration 
Basal metabolic rati! 

protein -bound Iodine In 
Bases, acids and buffers 

primary standards 
Barbiturates 

paper chromatography 

% (table) 
Beer' a Law 
Bibliography 

Bicarbonate , in acid-base balance 
Bilirubin 

types 

micro -method 
Biuret methods, protein 
"Blank", photometric 
Blindness, due to methyl alcohol 
Blood, coagulation of 
Blood collection 

anti -coagulants, various 

capillary 

centrifugation 

changes upon storage 

choice of sample 

errors in sampling 

freezing of serum 

hemolysis, prevention of 

plasma 

preparation of, for analysis 

serum 

"Vacutainer" tubes 

venous 
Bloor method, for cholesterol 
Bodansky, phosphatase unit 
Bone disorders 

phosphatase in 
Boutwell, urea method 
Boyle's Law 
Bromide 
Bromsulfalein 

clearance 

extraction analysis 

interferences 

tolerance 





5 


Buffers, acids and banes 




39 


7,10 


calculation it 




37 






table of 




47 




193 










39 


Calcium 


118, 


127 




22 


balance uludy, diet (Aub) 




125 




287 


balance lest 




123 




292 


In coagulation 123, 


,243, 


324 




293 


in disease states 


122, 


305 


49,55 


Calculations 








341 


colorimetric 




50 




321 


gasometrlc 


61 


,129 


107 


,304 


photometric 




57 


107 


,187 


volumetric 




32 




109 


Calculi, urinary 




300 


236 


,237 


Calibration, of photometer tubes 


i 


330 




56 


Capillary, blood collection 




70 




92 


Carbon Dioxide 


129 


,304 




324 


acid-base balance 


129 


,321 




70 


acidosis and alkalosis 




130 




73 


volumetric correction table 




132 




70 


chloride shift 




134 




71 


collection for 




129 




74 


Hamburger effect 




134 




72 


manometric correction table 




138 




74 


Carbon monoxide 




140 




75 


Carcinoid 




186 




71 


Carotenemla 




187 




72 


Cephalin flocculation 


144 


,309 




72 


Central tendency 




317 




72 


Centrifugation of blood 




71 




75 


Changes during storage 








70 


blood 




74 




152 


urine 




68 




225 


Charles' Law 
Chloride 




62 
304 


221 


,227 
264 


in acid-base balance 
argentimetric methods 




321 




61 


adsorption indicator 




147 




110 


thiocyanate indicator 




148 


112 


,304 


mercurimelric method 




145 




115 


protein effect in 




146 




114 


shift (Hamburger effect) 




134 




112 


Cholesterol methods 


152 


,154 




112 


in disease states 




154 



351 



CholinoHturaHO 


165 


"Christmas" disease 


326 


Chromatography 




of barb it urates 


292 


of amino acids 


96 


Circulating a ntl -coagulants 


328 


Cirrhosis 




coagulation defects 


326 


thymol turbidity In 


257 


transaminase in 


263 


Citrate, as ant 1 -coagulant 


73 


intoxication 


123 


Classification 




of esterases 


165 


of fat in feces 


170 


Cleaning of volumetric glassware 


12 


Clearance 




creatinine 


163 


urea 


310 


uric acid 


310 


Clinical Interpretation (table) 


305 


Coagulation of blood 


324 


calcium in 


123 


defects and diseases 


326 


schema (Owren) 


324a 


synonymy of terms in 


326 


Coeliac disease 




fat in feces in 


173 


Collection 




of blood 


70 


for carbon dioxide 


129 


of feces 


68 


of urine 


67 


Colorimeter, use of 


51 


Colorimetrlc analysis 


48 


calculations, problems 


36 


pH determinations 


44 


limitations of 


46 


techniques, apparatus and 




calculations 


48 


Congo red lest 


158 


Control procedures and analyses 


315 


types of variability 


316 


Conversion formulae-temperature 


63 


Convertln 


324 



Cupruporphyrln 233,235 

CuMlinini. 159,160 

clearance 163 

simple blood test for 295 

specificity 159 

Creatine, and creatine -phosphate 162 

Cretinism 

protein-bound iodine In 193 

Cunning's syndrome 

glucose tolerance In 184 

Cystic fibrosis of pancreas 

sweat electrolytes In 151 

Cystinuria, amino acids In 96 

Dal ton's law 64 

Definitions 

acids, bases and buffers 39 

volumetric 11 

Dehydration 304 

acid-base equilibrium and 322 

Density, optical vs. transmittance 

(table) 336 

Deproteialzation and principles of 76 

of serum 78 

Diabetes mellltus 304 

acetone in 84 

acid-base equilibrium In 322 

cholesterol in 154 

glucose In 182 

glucose tolerance In 184 

Diabetes Insipidus 

Kepler-Power water test In 86 

Diarrhea 304 

acid -base equilibrium in 322 

Dicoumarol 328 

poisoning 327 

Dispersion 317 

Drying of volumetric glassware 131 

Edema 304 
EDTA (ethylenedlaminetetraaceiic 

acid) as anti-coagulant 73 
Ehrllch's reaction 

for porphobilinogen 229 



352 



Kleclrolyles 

conversion factors (table) 

tn sweat 
Electromelric methods for pit 
Errors In blood collection 
Esterase 

classification 
Estrogens 
Ethyl alcohol 

"Factor" use of 

tn secondary standard 8 
Fanconl syndrome , amino acids In 
Fat tn feces 

classification and diseases 
Feces collection technique 
Fibrin 
Fibrinogen 

methods 

deficiency 

and sedimentation rate 
Fibrinoklnaae 
Flbrlnolysln 

Filters, photometric, choice of 
Filtrates, probe in-free 
Flame photometry 
Fluoride, as anti-coagulant 

and preservative 
Folln-Wu, deproteinization method 
Follicle stimulating hormone (FSH) 
Formula 

for buffer pH calculations 

for colorimetric methods 

for photometric methods 
Freezing, of serum 

Oasometrlc analysis 
Gastro enteritis, fecal fat in 
Glass electrode, for pH 
Globulin, gamma 

thymol turbidity 

zinc turbidity 



304 


Olucouc in blood 


181 


322 


true blood «uf»ar 


182 


150 


simple blood Lout for 


295 


46 


variations In disease 


184 


74 


Glucose tolerance 


1B3 


164 


variations in disease 


184 


165 






166 


Haden, method of deproteinization 


77 


68 


Hamburger effect (chloride shift) 


134 




Hcdulin 


328 




Hemochromatosis 197 


,200 


30 


Hemoglobin 


304 


l 96 


Hemolysis, prevention of 


71 


1T1 


Hemophilia 


326 


174 


Hemorrhagic disease Of new-born 


327 


68 


Hemosiderosis 




324 


Iron -bin ding capacity in 


200 


324 


Henry's law 


64 


175,177 


Heparin 74 


,328 


327 


Hepatic disease (see also Liver 




178 


disease and Liver function) 




326 


amino acids in 


270 


326,328 


cephalln flocculatlon in 


144 


54 


fibrinogen in 


178 


76 


hepato-cetiular 


304 


59,250 


phosphatase in 221 


,227 




urea in 


270 


73 


Hepatitis, acute infectious 




d 77 


Berum iron in 


197 


179 


porphyrins in 


236 




thymol turbidity tn 


257 


46 


zinc turbidity in 


273 


49 


"H" flocculatlon 




57 


as liver function test 


296 


75 


Hydrochloric acid 0, 1 N 


28 




Hydrogen electrode 


46 


61 


B-Hydroxy- butyric acid 




174 


acid-base equilibrium and 


322 


46 


5-Hydroxy-indolacetic acid 


185 




carcinoid, malignant 


186 


257 


Hy pe r i n sul inl s m , 




273 


glucose tolerance in 


184 



353 



Hypcrpn ralhy roidiam 




Karr urea method 




268 


phosphatase In 


221,227 


Kepler -Power water test 




65 


Hypertension 


304 


17-Kcto-ateroids 




201 


ubc of thiocyanates in 


256 


Kidney disease (see also Renal 






Hyperthyroidism 




disease and Henal function) 






creatinine in 


163 


creatinine In 




163 


protein -bound Iodine In 


1D3 


King -Armstrong, phosphatase unit 


218 


Hypoglycemia 


304 


King and Bain, chloride method 




149 


Hypoglycemic atonia 




Kjeldaht nitrogen 






glucose tolerance In 


184 


macro-method 




213 


Hypothyroidism 




micro-method 




215 


protein -bound iodine in 


143 








Icterus index 


187,299 


Laboratory tests, choice of 




304 


carotenemia 


1ST 


Lead 




284 


Indicators (table of) 


44 


Lipase 166 


,197 


.207 


Infarction, acute myocardial 




Llpemia 






transaminase in 


263 


cholesterol in 




154 


Infection, acute 




Lipid phosphorus 




228 


Iron-binding capacity In 


200 


Lipoid nephrosis 






Instruments, photometric 


68 


congo red test in 




158 


Insulin 




Liver disease (see also Hepatic 






glucose effect 


182 


disease) 






phosphate effect 


22T 


amylase in 




100 


resistance 


184 


cholesterol in 




154 


tolerance test 


184 


coagulation defects in 




326 


Intoxication, see also Toxicology 




esterase in 




166 


bromide 


111 


Iron-binding capacity in 




200 


ethyl alcohol 


80 


porphobilinogen in 




229 


carbon monoxide 


143 


transaminase In 




263 


citrate 


123 


zinc turbidity In 




273 


methyl alcohol 


92 


Liver function -tests and analyses 




salicylates 


249 


amino acids 




96 


sulfonamides 


264 


amylase 




100 


thlocyanatea 


266 


bilirubin 




107 


Iodine, protein -bound 


188 


bromsulfaleln 


112 


,116 


in various diseases 


193 


cephaUn flocculation 


144 


,309 


radio-active 


183 


cholesterol 


162 


,164 


Iron, serum 


198 


coagulation of blood 




324 


Iron binding capacity of serum 


198 


esterase 
fecal fat 




165 
173 


Jaundice 




fibrinogen 




176 


bilirubin in 


108 


"H" flocculation 




296 


cholesterol In 


154 


icterus index 


187 


,296 


fecal fat In 


173 


iron, serum 




186 






364 



Liver function-Tests and unulyscs (cont.) 
iron-binding capacity, scrum 198 



phosphatase 
porphyrins 
proteins, Berum 
prothrombin 
thymol turbidity 
transaminase 



urea 

Wilson's disease 

zinc turbidity 
Lead poisoning, porphyrins in 
Logarithms 

rules and uses Of 

table of 
Lubrication of stopcocks 



218,225 
230,308 
236,308 
240,324 
257 
258 
264,268,310 
88 



MacLaglan units, of turbidity 
Magnesium, methods 

in various diseases 
Malnutrition 

Manometric method, for CO„ 
Marking, for feces collection 
Mcllvaine standards for pH 
Mean 

Measurement, of urine volume 
Menopause, estrogens In 

follicle stimulating hormone in 
Mercury- 
Mercurlmetric method for chloride 

primary standard 
Methods of analysis 

colo rime trie 

gasometric 

requirements for clinical 

photometric 

volumetric 
Methyl alcohol 
Micro methods 

bilirubin 

cholesterol 

deprotelnization 
Molar gas volume 
Mucoviscidosis, sweat electrolytes 



273 
235 

41,42 

334 

IB 

330 

209,211 

210 

304 

137 

68 

45 

317 

67 

170 

179 

282 

146 

23 

3 

48 

61 

4 

53 

11 

91 



109 

167 

80 

66 

151 



Multiple myeloma 304 

calcium In 123 

Mumps, amylase in 100 

Muscular dystrophy, creatine in 163 

Myasthemla gravis, creatine in 163 

Myocardial infarction 

transaminase in 263 

Nephritis 304 

acid -base equilibrium In 322 

cholesterol in 154 

creatinine in 163 

Kepler -Power water teBt in 86 

phosphate in 227 

urea in 270 

Nesslerization 216 

Nitrogen determination 

macro-kjeldahl 213 

micro-kjeldahl 216 
non-protein nitrogen 215, 217 

Obstruction, biliary 

phosphatase in 221 

Obstruction, urinary, urea in 270 
Occlusion, acute myocardial 

transaminase in 263 

Occult blood 304 

Oliguria, urea in 270 
Optical deusity-transmittance 

table 336 
Oxalate, mixed 

anti-coagulant for hematology 73 
Oxidation-reduction 

primary standards 23 

Paget' s disease of bone 

phosphatase in 227 

Pancreatic adenoma, glucose in 182 

Pancreatic disease 

esterase in 165 

fecal fat in 174 

sweat electrolytes in 151 

Pancreatitis, acute 

amylase In 100 
lipase in 197 , 207 



355 



Paper chromatography 




of amino adits 


90 


of barbiturates 


292 


Parathyroid deficiency 




calcium in 


122 


pH, of blood 




acid-base balance and 


321 


carbon dioxide and 


130 


pH, definition 


40 


pH, determinations 




calculation by formula 


42 


buffers (table) 


47 


colorimetric methods 


44 


electrometric methods 


46 


scale 


40 


standards, Mcllvalne 


45 


primary 


24 



Phosphatase, acid and alkaline 218,225 

in various diseases 221,227 

tartrate sensitive 218 

"prostatic" fraction 221 

Phosphate, effect on calcium 122 

Inorganic 224 

method for magnesium 211 

lipid 228 

Photometric analysis, calculations 53 

problems 36 

flame photometry 59,250 

Pipets, precautions in use of 16 

Plasma, preparation of 72 

Plasma prothrombin time 240 

Plasma thromboplastin antecedent 

(PTA) 324,326 
Plasma thromboplastin component 

(PTC) 324 

Platelet factor 324 

Poisoning, treatment 275 

Poliomyelitis, porphyrinuria In 235 

Porphobilinogen 229 

Porphyrins, copro- and uro- 230 

Porphyria, acute 308,235 

Potassium 304 

in acid-base balance 321 

and sodium by flame photometry 250 

in sweat 151 



Potassium oxalate, anti-coagulant 73 

Potassium permanganate 0. 1 N 28 

Pregnancy, estrogens in 170 

magnesium, in toxemia of 210 

urea In 270 

Preparation, of blood for analysis 72 

Preservation, of blood 73 

of feces 69 

of urine 67 

Primary standards 20 

Pro-acce'erin 324 

deficiency 327 

Problems 

in buffers 37 

In colorlmetry 36 

in photometry 36 

in volumetric analysis 32 

Proconvertin 324 

deficiency 327 

Pro-fibrinolysln 326 

"Prostatic" acid phosphatase 218,221 

Prostatic carcinoma 221,227 

Prostatic hypertrophy 304 

Protein 304,308 

binding, of calcium 122 

chloride, effect on determination 146 

methods 

Protein-bound iodine 

Protein-free filtrates 

Prothrombin 

Prothrombin time 

conversion table 

time to per cent 
reagents, preparation of 
Pseudo-chollnesterase 
Pulmonary Insufficiency 

Reinach test for heavy metals 
Renal disease 

acid-base equilibrium in 

magnesium in 

urea In 
Renal function -teats and analyses 

acid-base equilibrium 321 

creatinine and clearance 159,160 



< 



236 


,237,239 




188 




76 




324,326 




240,248 




242,243 




245 




165 




304 


8 


277 




304 




322 




210 




270 



356 



Hen ill function-testa and sinalysea (cant.) 

creatinine unci clearance (cont.)235,30 5 

magnesium 210 

non-protein nitrogen 215,217 

phosphate 224 

urea and clearance 264,268,310 

uric acid and clearance 271,310 

Rheumatic fever, salicylates in 249 

nine turbidity in 
Rickets, calcium In 

phosphatase In 

phosphate In 
Salicylic acid 
Secondary standards 
Sedimentation rate 

and fibrinogen 
Serotonin 

5-Hydroxylndolacetlc acid 
Serum, collection 

deprotelnlzatlon 

freezing 
Serum prothrombin 
Scarlet fever, zinc turbidity in 
Schoenhelmer and Sperry 

cholesterol method 
Shank- Hoagland turbidity units 
Siliconizing, prothrombin tubes 
Slmmond's disease 

glucose tolerance In 
Simple blood test 

calibration table 

creatinine 

glucose 

"H" flocculatloa 

icterus Index 

sedimentation rate 
Slide rule, use of 
Sodium and potassium 

flame photometry 

In acid-base balance 
Sodium hydroxide 0.1H and 2. 5_N 
Sodium thiosulfate, 0.1M 
Solutions, preparation of 
Somogyi, deproteinisation method 



273 

123 
221,227 

227 

249,309 

27 

296,304 

178 

185,186 

72 

78 

75 

248 

273 

154 
330 
247 

184 

294 

297 

295 

295 

296 

296 

295 

333 

304 

250 

321 

28,30 

29 

19 

79 



Spina) fluid 312 

Sprue , fecal fat lit 174 

Standard conditions, gasamotrlc 61 

Standard deviation, statistics 317 

Standard reference texts 340 

Standards, plf, Mc livable 45 
Standards 

primary 20 

secondary 27 

Standardization, techniques of 22,28 

29,32 

Starch solutions, preparation of 31 

Statistics, terms and use of 317 

Steatorrhea, calcium in 123 

Sterol esterase 165 

Stock reagents (table) 27 

Stress, glucose in 182 

Sugar in blood 181, 304 

Sulfonamides 254,309 

Sulfuric acid, 2/3 N and 1/12 N 30 

Sulkowitch test, urine calcium 306 

Sweat electrolytes 150 

Synonyms In coagulation 326 



Tartrate sensitive phosphatase 
Temperature conversion formulae 
Testicular dyBtrophy 

17-ketoaterolds In 
Tetany, calcium in 
Thiocyanates 
Thrombin 
T h rombocytopenia 
Thromboplastin 
Thymol turbidity 

turbidity units 
Thyroid carcinoma and thyroiditis 

protein-bound iodine in 
Thyroid disease , cholesterol In 
Titan yellow, magnesium method 
Toxicology (see also Intoxication) 

arsenic 

barbiturates 

paper, chromatography 
qualitative 



221 
63 



206 
123 
256 
324 
326 
244, 324 
257,304 
329 



193 
154 
209 
274 
279 

292 
292 



357 



Toxicology (cont.) 




Vomiting 


304 


barbiturates (cont.) 




acetone in 


84 


quantitative 


287 


ncld-basc equilibrium in 


322 


Kf values (table) 


293 






lead 


284 


Water balance 


321 


mercury 


282 


Water, ionization of 


39 


Tromexan 


328 


total body 


256 


Transaminase 


258 


vapor pressure (table) 


65 


temperature correction table 


260 


Weights, care and Use of 


7 


Transmittance vs. optical density 




calibration 


10 


(table) 


336 


Wilson's disease, amino acids in 


96 


Turbidity standards 


329 






thymol turbidity test 


257 


Zinc turbidity (gamma globulin) 


273 


zinc turbidity test 


273 


turbidity units 


329 


Urea nitrogen, and clearance 304 


,310 






methods 264 


,268 






Uric acid, and clearance 


310 






method 


271 






sodium cyanide In 


272 






Urinalysis, use of 


304 






Urinary calculi, analysis 


298 






Urine 








exe ration of acid, effect of 


331 






ammonia, method for 


267 






collection, outline of 


67 






preservation 


6T 






Urobilinogen (and porphobilinogen) 


229 






Uroporphyrin 134 


,235 







< 



< 



"VacutaiwV tubes (blood collection) 76 
Van Slyke apparatus 

volumetric, for CO x 132a 
Van Slyke and Hawkins 

method for dap rote Inisatlon 78 

Venous blood collection 70 

Vitamin D deficiency, calcium in 122 

Vitamin K In coagulation 326 

Volume calibration of test tubes 332 

Volumetric analysis 11 

principles 17 

solution preparation 19 

Volumetric calculations, problems 32 



368 



( 



LONG TOM* OF TBI PERIODIC TABLE 





Part ad 


IA 


tu 






















mil 


ivb 


VB 


via 


ves 


O 






I 












i 


2 




1 


a 

LOW 
































H 

1.004 


at 

4,003 






1 


4 


5 


s 


7 


s 


9 


10 




1 


LI 
0.040 


t.oii 






















B 
10.81 


c 

12.01 


M 

14.006 



16.00 


F 
It. 00 


20. 161 






11 


11 


11 


14 


15 


10 


17 


16 




1 


13. HI 


Mi 
M.a 






















A] 
20.04 


Si 
U.OO 


P 
10.075 


1 

32.008 


CI 
3S.4S7 


A 
39.944 




m» 


IVA 


V*. 


VIA 


TEA 




tola 




a 


OB 






II 


» 


ti 


11 


13 


M 


» 


M 


2T 


28 


20 


30 


31 


32 


33 


34 


35 


36 




4 


K 


c* 


Ic 


Ti 


Y 


Cr 


Mb 


Fa 


Co 


Hi 


Cm 


Xa 


G» 


0* 


At 


i* 


Br 


Kr 






N.100 


♦e.M 


44,16 


47.00 


SO. OS 


01.01 


04.03 


U.OO 


66.04 


04.00 


63,54 


85. M 


80.72 


72.00 


74.91 


76.98 


"9.916 


83.90 




17 


» 


as 


40 


41 


42 


43 


44 


40 


40 


47 


48 


49 


50 


SI 


52 


53 


54 


& > 


n 


Or 


T 


Zr 


Hb 


MO 


Tc 


ItB 


b 


Pd 


Af 


Cd 


In 


5b 


5b 


T« 


I 


Xt 


t 





63.44 


IT. S3 


IS. 02 


01.22 


01.01 


15. IS 


0* 


101.7 


102.01 


10S.7 


If 660 


112.41 


114.76 


116.70 


121.73 


127.81 


126.91 


131.3 






Si 


ss 


37 -ti 


71 


TI 


74 


78 


78 


77 


78 


79 


60 


81 


82 


S3 


64 


S3 


66 




S 


C» 


BA 


La-Lu 


HI 


T» 


W 


JU 


Oa 


It 


Pt 


Au 


Hj 


Ti 


Pb 


Bl 


Po 


A: 


Ha 






112.11 


IjV.36 


fUre 
Earths 


170.0 


IIS. II 


113.01 


ISO. 31 


100.2 


103.1 


195,23 


197.2 


200.61 


204.30 


207.21 


209.00 


310 


210 


222 






17 


11 




00-102 




























1 




7 


Fr 

an 


21S. 35 




Acttablaa 




































17 


OS 


SB 


SO 


01 


S2 


S3 


84 


65 


68 


67 


66 


69 


TO 


71 








Rare Eartu 


La 


Co 


Fr 


rfd 


Pm 


Sis 


Ea 


Gd 


Tb 


Dy 


Ho 


Er 


T* 


Yb 


Lu 










138.92 


140.13 


140.92 


144.27 


14S 


150.43 


152.0 


136.9 


159.2 


162.46 


164.94 


167.2 


169.4 


173.04 


174.99 










SI 


10 


11 


02 


93 


04 


95 


96 


97 


98 


99 


100 


101 


102 










AeUaldaa 


Ac 


Th 


Pa 





Hp 


Pu 


Am 


Cm 


3k 


Cf 


El 


Fm 


Md 


No 












227 


232.12 


231 


236.07 


237 


242 


243 


243 


245 


248 


235 


252 


236 










INTKIINATIdhAI. ATOMIC WKICIITS 
I Will 






Actinium 

Aluminum 

Amrrlclum 

Antimony 

Arpon 

Araonlc 

Anlnltne 

Dnrliim 

Tin rk nil 11 in 

Beryllium 

R! Rnuilh 

Boron 

Bromine 

Cadmium 

Calcium 

CnUfornlum 

Cnrlxm 

Cerium 

Cesium 

Chlorine 

Chromium 

Cobalt 

Copper 

Curium 

Dyaproatum 

Einsteinium 

Erbium 

Europium 

Fermium 

Fluorine 

Franclum 

Gadolinium 

Gallium 

Germwilum 

Ootd 

Hafnium 

Helium 

Holmlum 

Hydrogen 

Indium 

Iodine 

Iridium 

Iron 

Krypton 

Lanthanum 

Lead 

Lithium 

Lutotlum 

Mn^ricBlum 

Mnngnnnae 

Mentlntovlum 





Atomic 


Atomic 


rmbol 


Number 


Woi K ht 


Ac 


80 


227 


Al 


13 


20.98 


Am 


05 


(243)* 


Sb 


51 


121.70 


A 


18 


39.944 


Aa 


33 


74.91 


At 


95 


(211) 


8a 


SB 


137.37 


Bk 


07 




Be 


4 


9. 013 


Bl 


S3 


209.00 


B 


5 


10.82 


Br 


36 


70.916 


Cd 


M 


112.41 


Ca 


20 


40.06 


Cf 


88 




C 


Q 


12.011 


Ce 


68 


140.13 


Ca 


56 


132.91 


CI 


11 


36. 457 


Cr 


24 


52.01 


Co 


27 


68.94 


Cu 


29 


63.54 


Cm 


9ft 


(245) 


Dy 


66 


162.81 


E 


09 


(254) 


Er 


68 


167.27 


Eu 


63 


162.0 


fm 


100 


(262) 


F 


9 


19.00 


Fr 


87 


(223) 


Od 


64 


167.26 


Oa 


31 


69.72 


Oe 


32 


72.60 


Au 


79 


197.0 


Hf 


72 


178.58 


He 


2 


4.003 


Ho 


87 


164.94 


H 


1 


1.0080 


i» 


49 


114.76 


I 


S3 


126.91 


*r 


77 


192.2 


Fe 


26 


W.85 


Kr 


36 


83.8 


La 


67 


138.92 


Pb 


82 


207.21 


14 


3 


6.940 


Lu 


71 


174.99 


Mg 


12 


24.32 


Mn 


25 


54.94 


Mv 


101 


(256) 







Atomic 




tjymbol 


Number 


Mnrcury 


Bg 


3D 


Molybdenum 


Mo 


42 


Ncodymlum 


Nd 


60 


Neon 


No 


10 


Neptunium 


Np 


03 


Nickel 


Nl 


28 


Niobium 


Nb 


41 


Nitrogen 


* 


7 


No helium 


No 


102 


Osmium 


Oa 


76 


Oxygen 


O 


8 


Pol Indium 


Pd 


46 


Phosphorus 


P 


16 


Platinum 


Pt 


78 


Plutonium 


Pti 


04 


Polonium 


Po 


84 


Potassium 


K 


19 


Praseodymium 


Pr 


59 


Promethium 


Pm 


61 


Protactinium 


Pa 


91 


Radium 


Ra 


88 


Radon 


Rn 


86 


Rhenium 


Re 


76 


Rhodium 


Rh 


45 


Rubidium 


Rb 


37 


Ruthenium 


Ba 


44 


Samarium 


Sm 


62 


Scandium 


te 


21 


Selenium 


8e 


34 


Silicon 


51 


14 


Silver" 


A* 


47 


Sodium 


Ha 


11 


Strontium 


Er 


38 


Sulfur 


S 


16 


Tantalum 


Ta 


T3 


Technetium 


re 


43 


Tellurium 


Te 


62 


Terbium 


ib 


65 


Thallium 


Tl 


51 


Thorium 


Th 


00 


Thulium 


Tm 


69 


Tin 


Sn 


60 


Titanium 


Tl 


22 


Tungsten 


W 


74 


Uranium 





92 


Vanadium 


V 


23 


Xenon 


Xe 


94 


Ytterbium 


Yb 


70 


Yttrium 


Y 


39 


Zinc 


Zn 


30 


Zlrcor.Ium 


Zr 


40 



Atomic 

Wc' ;ht 



200. 

95. 

141. 

21. 
(23?) 

68. 

92. 

14. 

(253) 

190. 

16 
108. 

30. 

195. 

(242) 

210 

39. 
140. 
(145) 
231 
22S. 
222 
186 
102 

85, 
101. 
150. 

44. 

78. 

28. 
IC7. 

22. 

87. 

32. 
180. 
(99) 
127. 
158. 
204. 
232. 
168 
118. 

47 
183 
238. 

50. 
131. 
173. 

88. 

65. 

91. 



01 

or, 

27 
183 

71 
9 1 
0D8 



7 

975 

09 



100 

92 



05 

32 

91 

48 

1 

35 

96 

96 

09 

880 

091 

63 

066 

95 

SI 

93 
39 
Oil 
94 
70 
90 
6C 

vt 

30 
04 
92 

38 

K2 



* A value given In 'brackets denotes the mass number of (be Isotope of longest known haH-llfe. 
/ SynlhcslKod In 1957. 

360 



(. 



|I,S. li.lVKHNMKNI HIINIINli OHB't . WM 11 - <*»!»! 

943-501